Mechanically induced trapping of molecular interactions

ABSTRACT

The invention provides devices and methods for surface patterning the substrate of a microfluidic device, and for detection and analysis of interactions between molecules by mechanically trapping a molecular complex while substantially expelling solvent and unbound solute molecules. Examples of molecular complexes include protein-protein complexes and protein-nucleic acid complexes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 11/698,757, filed Jan.26, 2007, now U.S. Pat. No. 8,039,269, issued Oct. 18, 2011, and claimsbenefit of U.S. Provisional Application Nos. 60/762,330 and 60/762,344,both filed Jan. 26, 2006, and to U.S. Provisional Application Nos.60/880,156 and 60/880,209, both filed Jan. 11, 2007. The entire contentof each of these applications is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Work described herein has been supported, in part, by the Office ofNaval Research (ONR)—Space and Naval Warfare Systems Center (Grant No.N66001-02-1-8929; Subcontract Princeton 341-6260-515). The United StatesGovernment may have certain rights in the invention.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file 87159-822538_ST25.TXT, created onJan. 10, 2012, 1,156 bytes, machine format IBM-PC, MS-Windows operatingsystem, is hereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to novel microfluidic devices and use ofmicrofluidic devices for analysis of interactions between molecules(e.g., biomolecules and/or chemical compounds). The invention findsapplication in the fields of biology, chemistry, medicine andmicrofluidics.

BACKGROUND

Analysis of molecular interactions involving biomolecules, such asproteins, nucleic acids, and glycans, is central to understandingbiological processes and is a critical step in drug development.However, quantifying the affinity of molecular interactions is aconsiderable technical challenge. First, there are often a large numberof variables that govern any particular biological interaction.Therefore obtaining equilibrium dissociation constants, for example,requires one to perform dozens of assays as the concentrations ofvarious components are systematically varied, increasing the number ofmeasurements needed in an already logistically challenging process. Asecond and more fundamental problem is the fact that many molecularinteractions are transient in nature and exhibit nanomolar to micromolaraffinities, leading to rapid loss of bound material or little boundmaterial in the first place. These factors are problematic forhigh-throughput methods such as yeast two-hybrid and tandem affinitypurification mass spectrometry where transient interactions arefrequently missed. Protein-protein and protein-DNA binding microarrays(PBMs) are especially susceptible due to their stringent washrequirements, causing rapid loss of weakly bound material. Proteinarrays have been applied to quantify ligand-ErbB receptor interactionswith off-rates determined by surface plasmon resonance to be on theorder of 10⁻⁴s⁻¹. PBMs have been applied in a semiquantitative manner totranscription factor (TF) motif analysis for high affinity interactions,with off-rates on the order of 10⁻³ s⁻¹.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for selectively modifyingthe substrate in a unit cell of a microfluidic device by (i) masking afirst portion of the substrate by contacting the first portion with amoveable element of the unit cell; (ii) contacting non-masked regions ofthe substrate with a substrate modifying agent; (iii) depleting orremoving the substrate modifying agent; and (iv) unmasking the firstportion of the substrate. In one embodiment the movable element is adeflectable elastomeric membrane. In one embodiment the deflectableelastomeric membrane is free-standing. In one embodiment contact betweenthe membrane and the substrate occurs medially and extends radiallyoutward.

In one version of the method the substrate modifying agent covalentlymodifies the substrate. In one version the substrate modifying agentnoncovalently modifies the substrate. In one version the substratemodifying agent is a protein or a nucleic acid. In one version thesubstrate modifying agent is an antibody, a receptor, a fusion protein,a glycan, a lipid, or a carbohydrate. In one version the substratemodifying agent is conjugated to avidin or biotin. In one version thesubstrate has previously been selectively modified.

In one aspect the invention provides a method using a unit cell of amicrofluidic device, said unit cell comprising in a liquid environment:a substrate, a molecular complex comprising a first molecule immobilizedin a contact area of the substrate, and second molecule bound to thefirst molecule and thus indirectly bound to the substrate, and atrapping element that upon actuation contacts the substrate in thecontact area of the substrate. The method includes actuating thetrapping element causing it to contact the substrate in the contact areathereby physically trapping the first and second molecules bound to thesubstrate in the contact area while substantially expelling solvent andsolute molecules. In one embodiment, the method includes detecting thetrapped first and/or second molecules.

In one embodiment, the method includes the step of de-actuating thetrapping element and, optionally, detecting the first and/or secondmolecules after deactuation.

In one embodiment, prior to de-actuating the trapping element, theliquid environment in the unit cell is changed. In one embodiment, priorto actuating the trapping element the complex is contacted with a thirdmolecule and the effect of the third molecule on formation ordissociation of the complex is determined. In various illustrativeversions of the invention, the first molecule is an antibody and thesecond molecule is an antigen or the first molecule is a protein and thesecond molecule is molecule bound by the protein.

In one aspect the invention provides a method using a unit cell of amicrofluidic device, said unit cell comprising in a liquid environment asubstrate, a first molecule immobilized in a contact area of thesubstrate, a second molecule, a movable element that upon actuationcontacts the substrate in the contact area of the substrate, where themethod includes actuating the movable element causing it to contact thesubstrate in the contact area thereby physically trapping the firstmolecule and any second molecules bound to the first moleculesubstantially expelling solvent and unbound second molecules. In oneembodiment, the method includes the step of de-actuating the movableelement. In one embodiment, the method is carried out on at least 100unit cells of a microfluidic device, and each of the 100 unit cellscomprises a different first molecule and/or a different second moleculeand/or optionally a different third molecule.

In one aspect, the invention provides a method of fabricating amicrofluidic device by i) positioning an elastomeric block comprising aplurality of chamber recesses and a solid support comprising amicroarray of discrete reagent-containing regions so as to align eachreagent-containing region with a recess; ii) adhering the block to thesolid support so as to produce a plurality of chambers containingreagents wherein each reagent-containing region contains two or morediscrete subregions, each containing a different reagent. In one aspect,the solid support is epoxy-functionalized glass. In one variousillustrative embodiments i) the microarray has a density of 100 or morediscrete regions per cm² or has a density of 1000 or more discreteregions per cm²; ii) the microarray comprises 10 or more differentreagents or 100 or more different reagents or 500 or more differentreagents. In some embodiments the reagents are proteins, nucleic acids,or small organic molecules.

In one aspect, the invention provides a microfluidic device comprising aplurality of unit cells, each unit cell comprising a microfluidic flowchannel having a substrate, a microfluidic chamber overlying the flowchannel, wherein said channel and said chamber are separated byelastomeric membrane and wherein an increase in pressure in the chambercauses the membrane to deflect into the channel and contact thesubstrate of the flow channel; and a second chamber in fluidiccommunication with the flow channel comprising a reagent in dry formdisposed on a reagent-containing region of the substrate wherein atleast 100 unit cells of the device each contains a different reagent,different amounts of a reagent, or a different combination of reagents.

In one aspect, the invention provides a microfluidic device comprising aplurality of unit cells, each unit cell comprising: a flow channelhaving a substrate, a membrane actuator chamber overlying the flowchannel and separated from the flow channel by an elastomeric membraneand, where an increase in pressure in the chamber causes the membrane todeflect into the channel and contact the substrate of the channel; and athird chamber in fluidic communication with the flow channel; each firstchamber is in fluidic communication with a first chamber in two adjacentunit cell(s), a valve that can be closed to fluidically separate thefirst and third chambers, a valve or valves that can be closed tofluidically separate the first chamber from first chambers in adjacentunit cells.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1D show a close-up view of a unit cell with control linesfilled with colored food dyes and empty flow layer. The blue controlline [b] creates the button membrane [bm] shown in the open (FIGS. 1Aand 1C) and closed (FIGS. 1B and 1D) configurations. Panels C and D showa schematic of a cross-section through the correlating image along thearrow-demarcated black lines. FIG. 1E shows one alternative design of atrapping element. Flow channels (red/gray) and control channels (crosshatched) are shown. White rectangles are “posts.” Closing the valvesmarked with X defines the unit cell.

FIGS. 2A-2D show a schematic of the surface chemistry that was generatedon the device as well as the process of protein synthesis, capture andMITOMI. Boxes indicate fluorescently labeled molecules. MAX isoA=fluorescein label; template=Cy3 label and Ebox=Cy5 label. FIG. 2Ashows the final surface chemistry just prior to introduction of the invitro transcription/translation reagents. Each grey block represents amonolayer consisting of the indicated molecule. FIG. 2B provides theprocess of protein synthesis using the deposited linear expressiontemplates. The synthesized MAX iso A protein diffuses to the antibodycoated surface and is pulled down via its N-terminal 6×Histidine tag.The free Ebox DNA molecules, introduced with the ITT mix, are recognizedby MAX iso A and likewise pulled down to the surface. In FIG. 2C MITOMIis performed by closure of the button membrane, trapping any boundmaterial and expunging any unbound material (corresponding image: FIG.3B). FIG. 2D shows the final state of the device after the last PBS washremoving any unbound material from the adjacent material (correspondingimage: FIG. 3C).

FIGS. 3A-3C shows testing the dependence of the actual spot size onmembrane diameter. FIG. 3A shows an Autocad diagram of a section of theactual device. Here 5 unit cells with different membrane diameters areshown. Heavy lines indicate control lines and light lines indicate flowlines. Membrane diameters are 180 μm on the far left decreasing to 100μm in 20 μm steps. The actual device has one additional unit cell with a80 μm membrane. FIG. 3B shows the fluorescence [seen as dark areas] ofCy5 labeled DNA templates filled in the flow channel. The membranes havebeen closed trapping DNA bound by a surface bound transcription factor.Note the halo of low intensity around the spots, indicative of lownon-specific binding of templates due to the membrane action. FIG. 3Cshows the same area of the device as FIG. 3B after flushing the flowchannel with PBS with the membranes remaining closed to prevent loss ofbound material.

FIG. 4 shows the relationship of spot size dependence and membranediameter.

FIG. 5 shows differences in apparent pull-down and trapping of freeEbox-DNA carrying various recognition sequences by MAX iso A with a6×His tag (open symbols) and non-tagged (closed symbols).

FIG. 6A shows a design drawing of the microfluidic device. Red [dark]and blue [light] lines represent control and flow channels,respectively. The device contains 2,400 unit cells controlled by 7,233valves (scale bar=2 mm). FIG. 6B shows an optical micrograph of threeunit cells. Control channels are filled with colored food dyes forvisualization. Each unit cell consists of a DNA chamber aligned to amicroarray spot, and a detection area. The valves colored green (marked“cv”) control access to the DNA chambers while the valves colored orange(“o”) compartmentalize the unit cells. The button membrane representsthe area where detection takes place (scale bar=150 μm). FIG. 6C shows aschematic outline of the approach. First a microarray of target DNAsequences is spotted onto an epoxy slide. The microarray is then alignedand bonded to a microfluidic device. Next the necessary surfacechemistry is prepared, followed by in situ synthesis of TF and detectionof interactions by MITOMI. FIGS. 6D-6F provide a schematic of theprocess of MITOMI. The gray structure at the top of each panelrepresents the deflectable button membrane that may be brought intocontact with the glass surface (“substrate”). FIG. 6D shows that His5tagged TFs are localized to the surface and TF-DNA binding is in asteady state. FIG. 6E shows that the button membrane is brought intocontact with the surface, expelling any solution phase molecules whiletrapping surface bound material. FIG. 6F shows that unbound material notphysically protected is washed away and the remaining molecules arequantified. FIGS. 6G-6I are fluorescent intensity maps of target DNAconcentration, corresponding to the states schematically shown in FIGS.6D-6F (scale bar=50 μm).

FIG. 7 illustrates three approaches to printing micro-arrays for usewith microfluidic devices. Circles indicate array spots and the label(A, B, 1, 2, alpha, beta) identifies the contents of the spot. Arrowsindicate the preferred direction in which spots are deposited (onlycertain arrows are shown). FIG. 7A depicts a standard micro-array whereeach spot is unique and originates from a unique solution. FIG. 7B showsa co-multispotted pattern in which over three rounds a three dimensionalmatrix is generated. First columns are spotted with the solutions A andB respectively, followed by spotting of solutions 1 and 2 in therespective rows directly on top of the previously spotted solutions. Inthe third round solutions alpha and beta are spotted. FIG. 7Cillustrates “neighbor multispotting.” The array in FIG. 7C is similar tothat in FIG. 7B, except that spots are placed adjacent to, rather thanon top of, one another.

FIG. 8A shows an overview of the experimental approach starting with aplain epoxy substrate to be spotted with 2400 spots of a target DNAlibrary. The finished microarray is then aligned and bonded to one ofour microfluidic devices after which the surface is prepared, proteinsynthesized and MITOMI performed. FIG. 8B is a micrograph of one of themicrofluidic unit cells, shown here again for reference. The dashedlines show which regions of the unit cell are schematically depicted inFIGS. 8C-8L. FIG. 8C: Before any fluid is introduced into the device thechamber valve (denoted “cv” and colored green in FIG. 8B) is closed toprevent flooding of the DNA chamber. FIG. 8D: Next biotinylated BSA isintroduced into our device which covalently bonds to the epoxyfunctional groups, both activating (via the biotin moieties) andpassivating (epoxy groups) the surface. FIG. 8E: A solution ofneutravidin is introduced forming a monolayer on top of the biotinylatedBSA layer. FIG. 8F: The “button” membrane is closed to protect thedetection area from passivation via biotinylated BSA which passivatesall accessible surface area. FIG. 8G: Any unbound biotinylated BSA ispurged before the “button” membrane is opened again allowing access tothe neutravidin surface below to which a biotinylated penta-histidineantibody is attached, concluding the surface derivatization. FIG. 8H:ITT programmed with linear expression template is introduced into thedevice and allowed to flood the DNA chamber causing the solvation of thestored target DNA. Transcription factor is being synthesized and ispulled down to the surface by penta-histidine antibody. FIG. 8I: Thesynthesized transcription factors functionally interact with thesolvated target DNA pulling it down to the surface as well. FIG. 8J:After 60-90 min the “button” membrane is closed again mechanicallytrapping any molecular interactions taking place on the surface allowingall solution phase molecules to be washed away without loss of surfacebound material FIGS. 8K-8L.

FIG. 9A shows a two step PCR method for generating linear expressiontemplates. FIG. 9B shows the 5′ and 3′ UTR sequences added by the 2 stepPCR method. Sequence legend: 5′ UTR sequence (SEQ ID NO:1); 3′ UTRsequence (SEQ ID NO:2). All regions are annotated and all primingsequences are in red. The start and stop codons are colored green. Theentire 5′ and 3′ UTRs are added by the 5′ extension and 3′ extensionprimers respectively except for the start and stop codons.

DETAILED DESCRIPTION

Definitions

As used herein, the term “microfluidic” device has its normal meaning inthe art and refers to a device with structures (channels, channels,chambers, valves and the like) at least some of which have at least onedimension on the order of tens or hundreds of microns. In general, atleast one structure of the device has dimension(s) below 1000 microns.

As used herein, “elastomeric” has its normal meaning in the microfluidicarts. Elastomers in general are polymers existing at a temperaturebetween their glass transition temperature and liquefaction temperature.See Allcock et al., Contemporary Polymer Chemistry, 2nd Ed. Elastomericmaterials exhibit elastic properties because the polymer chains readilyundergo torsional motion to permit uncoiling of the backbone chains inresponse to a force, with the backbone chains recoiling to assume theprior shape in the absence of the force. In general, elastomers deformwhen force is applied, but then return to their original shape when theforce is removed. The elasticity exhibited by elastomeric materials maybe characterized by a Young's modulus. Elastomeric materials having aYoung's modulus of between about 1 Pa-1 TPa, more preferably betweenabout 10 Pa-100 GPa, more preferably between about 20 Pa-1 GPa, morepreferably between about 50 Pa-10 MPa, and more preferably between about100 Pa-1 MPa are useful in accordance with the present invention,although elastomeric materials having a Young's modulus outside of theseranges could also be utilized depending upon the needs of a particularapplication. Given the tremendous diversity of polymer chemistries,precursors, synthetic methods, reaction conditions, and potentialadditives, there are a huge number of possible elastomer systems thatcould be used to make the devices of the invention. Common elastomericpolymers include perfluoropolyethers, polyisoprene, polybutadiene,polychloroprene, polyisobutylene, poly(styrene-butadiene-styrene),polyurethanes, and silicones, for example, orpoly(bis(fluoroalkoxy)phosphazene) (PNF, Eypel-F),poly(carborane-siloxanes) (Dexsil), poly(acrylonitrile-butadiene)(nitrile rubber), poly(I-butene),poly(chlorotrifluoroethylene-vinylidene fluoride) copolymers (Kel-F),poly(ethyl vinyl ether), poly(vinylidene fluoride), poly(vinylidenefluoride-hexafluoropropylene) copolymer (Viton), elastomericcompositions of polyvinyl chloride (PVC), polysulfone, polycarbonate,polymethylmethacrylate (PMMA), and polytertrafluoroethylene (Teflon),polydimethylsiloxane, polydimethylsiloxane copolymer, and aliphaticurethane diacrylate.

As used herein, “unit cell” refers to a combination of microfluidicstructural elements that is repeated many times (e.g., 48 to 10,000times, 100 to 5,000 times, or 250-2500 times) in a microfluidic device,where unit cells can operate simultaneously to carry out a function in ahighly parallel manner.

As used herein, “substrate” refers to a surface in a chamber or channelin a microfluidic device. Usually a chamber or channel can be defined byreference to substantially planar surfaces (e.g., floor, ceiling, andwalls) and “substrate” refers to a particular planar surface, e.g., the“floor.” More particularly, “substrate” refers to an exposed surface andmay change over time. For example, in a microfluidic chamber in whichone surface is formed by a solid support (e.g., an epoxy glass slide)coated with BSA, the substrate is the BSA layer. If this substrate isuniformly derivatized with an NHS-ester biotin to produce a biotinlayer, the substrate is the biotin layer. If one portion of the biotinlayer is derivatized by binding streptavidin, the substrate is thebiotin layer in some regions (those not derivatized) and thestreptavidin layer in other regions (derivatized regions). In a unitcell, a “chamber substrate” refers to the substrate in a chamber, a“channel substrate” refers to the substrate in a channel, and a “unitcell substrate” refers to a substrate in any region of the unit cell.

As used herein, a trapping element such as a trapping membrane or buttonmembrane is “free-standing” if, when actuated (i) at least some portionof the membrane is in contact with the substrate of a flow channel and(ii) the membrane does not contact the side walls of the flow channel.

As used herein, the verb “mask” refers to the process of physicallycontacting and covering a portion of a unit cell substrate so as toexclude a substrate modifying agent and prevent the covered portion frombeing modified by the substrate modifying agent.

As used herein, the term “elastomeric block” refers to the elastomericportion of a microfluidic device made using multilayer soft lithographytechniques, which has not yet been adhered to a solid support (orsubstrate). The elastomeric block contains a plurality of “chamberrecesses” that, upon attachment of the solid support form chambers inwhich the solid substrate forms one surface (e.g., the “floor”).

As used herein, the term “flow channel” refers to a microfluidic channelthrough which a solution can flow. The dimensions of flow channels canvary widely but typically include at least one cross-sectional dimension{e.g., height, width, or diameter) less than 1 mm, preferably less than0.5 mm, and often less than 0.3 mm. Flow channels often have at leastone cross-sectional dimension in the range of 0.05 to 1000 microns, morepreferably 0.2 to 500 microns, and more preferably 10 to 250 microns.The channel may have any suitable cross-sectional shape that allows forfluid transport, for example, a square channel, a circular channel, arounded channel, a rectangular channel, etc. In an exemplary aspect,flow channels are rectangular and have widths of about in the range of0.05 to 1000 microns, more preferably 0.2 to 500 microns, and morepreferably 10 to 250 microns. In an exemplary aspect, flow channels havedepths of 0.01 to 1000 microns, more preferably 0.05 to 500 microns,more preferably 0.2 to 250 microns, and more preferably 1 to 100microns. In an exemplary aspect, flow channels have width-to-depthratios of about 0.1:1 to 100:1, more preferably 1:1 to 50:1, morepreferably 2:1 to 20:1, and most preferably 3:1 to 15:1, and often about10:1. A flow channel need not have a uniform width along its length and,as described below, may be wider in the region in which the detectionarea is situated in order to accommodate a trapping membrane or othertrapping element. For example the portion of the channel substrate thatcontains the detection region may be widened (i.e., wider than otherportions of the flow channel) and/or rounded (see FIG. 1A and FIG. 3).Such a flow channel can be referred to as a “bulged flow channel.” Theportion of the channel substrate that contains the detection region canbe widened and rounded, as noted, or have a different shape.

General Materials and Fabrication Methods

Materials and methods for producing a variety of microfluidic devicesare known in the art. For illustration and not limitation, a briefdiscussion of useful methods is provided infra in the section entitled“General materials and fabrication methods.”

Introduction

In one aspect the invention provides a microfluidic device having aplurality of unit cells, where each unit cell contains (i) amicrofluidic flow channel and (ii) a moveable element that uponactuation can contact and mask a portion of the channel substrate.

In a second, related, aspect, the invention relates to methods for“surface patterning” a substrate of a microfluidic device by (i) maskinga first portion of the substrate of a microfluidic channel; (ii)contacting non-masked regions of the channel with a substrate modifyingagent; (iii) and unmasking the first portion. This results in asubstrate that has both modified and unmodified regions and may be usedto, for example, bind macromolecules to predetermined regions of amicrofluidic substrate.

In a third, related, aspect, the invention relates to a method fordetecting molecular interactions, referred to as “mechanically inducedtrapping of molecular interactions” or MITOMI. In MITOMI, amicro-mechanical membrane makes contact with a surface, physicallytrapping all surface bound molecules in the contact area, whileexpelling any unbound solvent and solute molecules. The trappedmolecules may then be directly observed and/or tested against othercompounds subsequently introduced into the system. The principal idea ofthis approach, the physical trapping of molecular interactions, isportable and thus applicable to a wide variety of systems and methodsinvolving interactions between any kind of molecule. In someembodiments, a micro-mechanical membrane makes contact with a surfaceupon pressure dependent deflection (i.e., a pressure-actuateddeflection).

In one example, MITOMI is used for fluid exchange in a microfluidicchannel having an immobilized molecular complex in which one member ofthe complex is bound directly to the substrate and a second member ofthe complex is bound to the first member. Fluid exchange is accomplishedwithout loss of bound material. Trapping of bound material protects itfrom subsequent wash and fluid exchange steps allowing backgroundmolecules to be removed and secondary reagents to be introduced into thesystem without disturbing the trapped material, allowing highlysensitive secondary assays such as ELISAs to be performed on thisplatform.

In one example, MITOMI is used to detect or characterize a molecularinteraction for example (but not limited to) specific binding, bindingaffinity, association or disassociation rates and the effect of onemolecule on an interaction between two other molecules.

Microfluidic Devices

MITOMI, surface patterning, and fluid exchange according to the presentinvention can be carried out using devices made using a wide variety ofmaterials and with a variety of designs. In this section, forillustration and not for limitation, exemplary devices (which areparticularly adapted for these processes) are described. Forconvenience, microfluidic devices of the invention can be referred to as“MITOMI devices,” although, as will be apparent from the discussionbelow, the devices described herein may be used for surface patterningand other uses in addition to MITOMI.

In one aspect the invention provides a MITOMI microfluidic device havinga plurality of unit cells, where each unit cell contains (i) amicrofluidic flow channel and (ii) a moveable element (a “trappingelement”) that upon actuation can contact and mask a portion of the flowchannel substrate.

The flow channel may have a variety of shapes and configurations. In oneembodiment the contact area is in a bulged flow channel.

The portion of the channel substrate that is contacted by the trappingelement is called the “contact area” or “mask area” or “detection area.”These terms are used interchangeably and refer to the same area of thesubstrate in different contexts: the area contacted by the trappingelement, which area is masked (preventing access by a substratemodifying agent) and where detection of trapped molecules occurs inMITOMI.

The trapping element may be a deflectable membrane or a non-membranestructure that can be actuated to contact a channel substrate (e.g., anelastomeric pad on a micromechanical extension arm). When the trappingelement is in the form of a deflectable (e.g., elastomeric) membrane itmay be free-standing or not free-standing. In one embodiment thetrapping element is a free-standing deflectable membrane and is called a“trapping membrane”. A generally circular trapping membrane can becalled a “button membrane” or “button.” In one embodiment the trappingmembrane is elastomeric.

Movement of the trapping element is controlled by an actuator. Movement(i.e., deflection) of a deflectable membrane, such as a trappingmembrane, can be controlled by a membrane actuator.

Microfluidic devices for use according to the invention may befabricated using any of variety of methods, materials andconfigurations. In a particular embodiment the device is made using thetechniques of multilayer soft lithography (MSL) and is referred to as an“MSL-type” device. To illustrate the invention, an MSL-type device withtrapping membrane, is discussed in detail in subsection (a), infra.Alternative embodiments are then discussed in subsection (b), infra.

a) MSL-Type MITOMI Device

In one embodiment the MITOMI device is made using elastomeric materialsusing MSL fabrication techniques. Elastomeric devices made using MSL arewell known, and familiarity with such devices by the reader is assumedin the description herein. (Additional guidance can be found in thescientific and patent literature, including references provided in thesection “General Materials and Fabrication Methods” infra.) Briefly,MSL-type devices typically include (i) a flow layer, in which aresituated flow channels through which liquids (including liquidscontaining analytes and reagents) can be transported; (ii) a solidsupport forming a surface (e.g., floor) of flow channels; and (iii) acontrol layer in which are situated control channels that overlay andintersect flow channels. At regions in which a control channel in thecontrol layer overlies a flow channel in the flow layer, the lumens ofthe flow channel and control channel are separated by a thin membrane ofelastomer. Increasing pressure in the control channel causes the thinmembrane to be deflected into the flow channel, thereby acting as avalve that blocks flow of liquid through the channel. Reducing thepressure in the control channel allows the membrane to retract out ofthe flow channel, opening the valve and allowing fluid to pass through.

The MSL-type MITOMI device also contains a flow layer, a control layerand a solid support and has specialized features for adapted for MITOMIand surface patterning. In one embodiment the MSL-type MITOMI device hasnumerous (e.g., 48 to 10,000 or more) unit cells having the followingbasic components:

-   -   i) a microfluidic flow channel situated in the flow layer, one        surface of the flow channel being the substrate surface;    -   ii) a membrane actuator chamber in the control layer adjacent to        (e.g., overlying) the flow channel;    -   iii) an elastomeric trapping membrane separating the membrane        actuator chamber and the microfluidic flow channel.        In this embodiment the membrane actuator chamber is connected to        a control channel by a thin via so that the chamber can be        hydraulically or pneumatically pressurized. Pressurization of        the chamber causes its floor (which in effect is the membrane        separating the chamber from the flow channel) to collapse and        ultimately make contact with, or mask, the channel substrate. As        alluded to above, this process can be used for masking to        produce a substrate with both modified and unmodified regions,        to trap, or protect, bound material for assays and analysis, and        the like.

In certain respects, although they have very different functions, thetrapping membrane in this embodiment is structurally similar to amonolithic valve because both are elastomeric membranes deflected into aflow channel. However, the trapping membrane in relation to the flowchannel is structurally distinct from a valve in one or more (dependingin the design of the device) of the following respects:

-   -   1. The trapping membrane is a free-standing membrane, i.e., when        actuated it contacts the channel substrate without contacting        the sides of the channel; and/or    -   2. The trapping membrane when fully actuated does not block flow        of fluid through the flow channel; and/or    -   3. The trapping membrane when actuated contacts a circular area        of the flow channel substrate; and/or    -   4. The trapping membrane contacts the flow channel substrate in        a bulged region of the flow channel; and/or    -   5. The trapping membrane does not block passage of particles,        such as a particle having a diameter of 5 to 50 microns, such as        more than about 5 microns, such as more than 10 microns, such as        more than 20 microns, such as more than 30 microns.        Particular trapping membranes can be characterized by any one or        combination of the properties listed above.

FIG. 1 illustrates a unit cell of an illustrative MITOMI device. Theactuator chamber is connected to a control channel on the same layer asthe chamber itself by a thin via so that the button chamber can behydraulically or pneumatically pressurized. FIGS. 1A to 1D illustratethe substrate (“glass”), flow layer containing a bulged flow channel, acontrol layer containing a membrane actuator chamber overlying the flowchannel, and a trapping membrane separating the actuator chamber andflow channel. FIGS. 1A and 1B are micrographs showing the trappingmembrane in an open (1A) and closed (1B) position. The round chambershown in the bottom half of FIGS. 1A and 1B is a reagent chamber, whichis discussed infra. In one design the actuator chamber is a smallercircular chamber aligned above the bulged flow channel. The circularchamber is connected to a channel on the same layer as the chamberitself by a thin via so that the chamber may be hydraulically orpneumatically pressurized. Pressurization of the chamber causes itsfloor, which in effect is the membrane separating the chamber from thebottom flow channel to collapse and ultimately make contact with thefloor of the flow channel beneath (see FIG. 1 panels C and D) It isnecessary that the valve geometry and closing pressure are adjusted sothat the valve membrane contacts only a portion of the substrate (i.e.,is not completely closed).

An increase in pressure in the membrane actuator chamber causes thetrapping membrane to deflect into the flow channel and make contact withthe channel substrate. In one embodiment, contact of the trappingmembrane with the floor occurs centrically (e.g., starts closing at themiddle of the membrane and then extends laterally outward as more of themembrane contacts the surface). As a consequence of this centric contactessentially no solvent molecules are trapped between the membrane andthe floor of the flow channel (detection area). This drastically reducesthe occurrence of non-specifically trapped solutes. In one embodiment acircular membrane closes radially (i.e., contact with the substratebegins at the approximate center of the membrane and radiates outward ina progressive manner).

In one design, the trapping membrane is generally circular (see FIG. 1)and can be referred to as a “button membrane” or “button.” Although acircular trapping membrane which contacts the channel floor centricallyworks particularly well in the applications described herein,non-circular button membranes also can be used, as discussed below. Thediameter of a button membrane is generally in the range 0.1 microns to1,000 microns, often in the range 1 micron to 500 microns and usually inthe range 10 microns to 500 microns.

Contact areas of various shapes (e.g., rectangular, circular, etc.) andsizes may be achieved by varying the geometry of the trapping membrane.geometries have been so far achieved (rectangular on the poster andcircular in the disclosure). The size of the area of contact between thetrapping membrane and substrate (the “contact area” or “mask area”) canbe precisely modulated by choice of button diameter and closingpressures (see FIG. 4 and Example 1). Typically the diameter of thecontact area is generally in the range 0.1 microns to 1,000 microns,often in the range 1 micron to 500 microns and usually in the range 5microns to 400 microns. It will be appreciated the size of the contactarea is generally a fraction of the size of the flow channel in which itresides. In some embodiments the diameter of a circular contact area isnot more than 75% of the width of the flow channel substrate at thelocation of the contact area, sometimes not more than 50% and sometimesnot more than 25%. Usually the contact area extends less that the widthof the flow channel. In some embodiments, the contact area extends notmore than 75% of the width of the flow channel, sometimes not more than50% and sometimes not more than 25% of the width of the flow channel. Incontrast, the area of contact of a conventional monolithic valve withthe substrate extends the entire width of the channel.

Closing pressures are generally in the range 12 and 18 psi, althoughother pressure ranges may be appropriate depending on configuration andmaterial. Useful closing pressures range from 0.1 psi to 100 psi,usually in the range 1 psi to 50 psi, and more often in the range 10 to20 psi.

In one embodiment of the invention having a generally circular trappingmembrane, the membrane actuator chamber is also generally circular, ormore precisely coin-shaped, with a diameter corresponding to that of thebutton membrane. It will be appreciated that the membrane actuatorchamber of a pressure-actuated MSL-type device is connected by a via tocontrol channels to actuate the membrane so that the membrane actuatorchamber can be pressurized causing the membrane to deflect. In generalthe membrane actuator chamber and button membrane have diameters smallerthan the underlying flow channel.

As noted, the shape and size of the flow channel near the contact areamay vary. For example, the flow channel may have any shape, so long asit is large enough for the deflectable membrane to deflect into thechamber and contact the substrate, preferably without contacting theflow channel walls. For example the flow channel may be circular,octagonal, rectangular and the like near the contact area.

The MITOMI contact area can be (and usually is) in a flow channel (whichmay be fluidically isolated). For illustration and not limitation, theisolated MITOMI contact area and reagent chambers often have volumes ofabout 0.5-1 nL. Exemplary isolated contact areas and reagent chambersmay have a generally circular footprint and have dimensions including adiameter of about 10 to about 1000 microns, e.g., from about 200-300microns, e.g., about 250 microns, and heights of from about 1 to about200 microns, e.g., about 5 to about 20 microns, e.b., about 10 microns.Contact areas and chambers having a non-circular shape may have similarvolumes.

Valves in the flow channel(s) may be used to fluidically isolate thecontact area. In one embodiment the flow channel has the configurationshown in FIG. 1. In this case three mechanical valves can be closed toisolate the contact area.

A microfluidic device having a chamber with an overlying elastomericmembrane that can be deflected into the chamber have been described formetering by volume exclusion. See U.S. 2002/0029814 para. [0454] et seq.This device contains a first elastomer layer with a control chamberoverlying a second elastomer layer with a dead-end reaction chamber. Thecontrol chamber overlies and is separated from dead-end reaction chamberby an elastomeric membrane. A reactant “x” may be introduced underpressure into dead-end reaction chamber. Increased control chamberpressure causes the elastomeric membrane to flex downward into reactionchamber, reducing by volume V the effective volume of reaction chamber.This in turn excludes an equivalent volume V of reactant from reactionchamber such that volume V of first reactant X is output from the flowchannel. These metering structures differ in function and structure fromthe unit cells of a MITOMI device. For example, the MITOMI contact areacan be (and usually is) in a flow channel (which may be fluidicallyisolated), while the metering structure is a dead-end reaction chamber.Further, when actuated the membrane of a metering structure does notcontact the chamber substrate.

Although not discussed above, it will be understood that the unit cellcan contain additional channels, valves, chambers, and othermicrofluidic elements, and the microfluidic device can includestructures and functional units other than the unit cells described.

Alternative Device Embodiments

As noted, one concept of the invention is the physical trapping ofmolecular interactions, and is applicable to a wide variety of systems.Essentially in order to perform MITOMI two prerequisites are required:surface bound molecules to be detected and a second structure that makescontact with the surface and in doing so expels any non-bound substancefrom the surface. In principal any two structures which can be broughtinto contact and fulfill above requirements are sufficient for thisapproach and any surface chemistry that is specific and materialcompatible may be used. Similarly, surface patterning requires asubstrate and a structure that masks the substrate and excludes asubstrate modifying agent.

Variations on the MITOMI device described above include (i) devices withan elastomeric trapping membrane, but not produced using MSL; (ii)devices in which actuation of the membrane is not effected by a changein pressure in a membrane actuator chamber; (iii) devices in which thetrapping membrane is not generally circular; (iv) devices in which thetrapping element is not free-standing; and (v) devices in which thetrapping element is not a membrane. FIG. 1E shows one alternative designin which the membrane is supported on either side by two “posts”situated in a bulged flow channel. In this design a MITOMI site isformed by the intersection of two flow channels. Within the bulged flowchannel two elastomeric “posts” or elongated “columns” (shown as whiterectangles in the figure) extend from the substrate to ceiling. Inresponse to an increase in pressure in the control channel (gray) theelastomeric membrane (“MITOMI valve”) is deflected into the flowchannels between the posts and contacts the substrate. Note that, incontract to the circular button membrane, which closes radially, thevalve starts closing in a line extending from the center of each postand then extends laterally outward as more of the membrane contacts thesurface.

In some embodiments, a hybrid device comprising an elastomericdeflectable membrane with remaining portions of the channels andchambers of the device being a composite structure made fromnon-elastomeric materials. See, e.g., U.S. Pat. Pub. No. U.S.2002/0029814.

In some embodiments of the invention, a button membrane is actuated by amechanism other than pressure. For example, use of electrostatic,electrolytic, electrokinetic, magnetic, thermal and chemical actuationsystems are also contemplated, as described in patent publicationU.S.2002/0029814 (paragraphs [0256] et seq.) in relation to actuation ofvalves. In some embodiments, devices in which the deflectable membraneis made using a thermoresponsive polymer gel.

In some embodiments of the invention, the trapping membrane is notgenerally circular and/or the trapping element is not free-standing. Forexample, in some embodiments, a conventional monolithic valve (formedfrom a flow channel and control channel) can be used for substratepatterning and MITOMI. In one version, using MSL with PDMS, a thin PDMSmembrane is generated between two channels formed in the PDMS andstacked and bonded on top of one another. In one version a valve thatacentrically blocks a flow channel, restricting flow on one side (i.e.,along one wall) of the channel but not completely shutting flow down andnot allowing flow along both walls of the channel (i.e., a “half-valve”)is used for MITOMI, surface patterning and fluid exchange. “Half valves”extend from one wall of the channel 10% to 80% of the way into thechannel (without touching the opposite wall), and can be made using a“blind” control channel that extends only part of the way across anelastomeric membrane over a flow channel.

In some embodiments, devices with nonelastomeric button membranes areused. In some embodiments, a nano electro-mechanical system (NEMS) ormicro electro-mechanical system (MEMS) is used in which a structureother than an elastomeric membrane is deflected into a microfluidicchamber to mask a portion of the substrate. Examples of such movableelements include silicon membranes or cantilevers, metal membranes andcantilevers. These membranes may be actuated by hydraulic, pneumatic oroptical pressure as well as magnetically, electrostatically ormechanically.

Reagent Chamber

In certain embodiments, the MITOMI unit cells includes a reagent chamber(called a “DNA chamber” in FIG. 6B) that is in fluidic communicationwith the flow channel (i.e., the detection area of the flow channel. SeeFIGS. 1A-B and FIG. 6B. The reagent chamber and the flow channel can befluidically isolated from each other (e.g., by closing a valve in thechannel that separates them). By opening the valve, reagents in thereagent chamber can be delivered to the flow channel and reagents in theflow channel can be delivered to the reagent chamber. In someembodiments a microfluidic pump is used to move fluids between thechambers. Importantly, in some embodiments micromechanical valves can beclosed so that the portion of the flow channel in which the detectionarea is situated is isolated from other unit cells and is incommunication with the reagent chamber; and micromechanical valves canbe closed so that the portion of the flow channel in which the detectionarea is situated is isolated from the reagent chamber.

In one aspect, a device of the invention contains a plurality of unitcells, each unit cell including a flow channel having a substrate and acontact area, a membrane actuator chamber overlying the contact area ofthe flow channel substrate, with the flow channel and chamber areseparated by elastomeric membrane so that an increase in pressure in thechamber causes the membrane to deflect into the flow channel and contactthe substrate; and a second chamber (reagent chamber) in fluidiccommunication with the flow channel. In an embodiment each reagentchamber is in fluidic communication via a flow channel with a detectionarea in the unit cell, each detection area is in fluidic communicationvia a flow channel with detection areas in two adjacent unit cells, andthe device includes valves that can be actuated to fluidically separatethe detection area in each unit cell from detection areas in other unitcells.

Reagents (solutes and reagents in solution or suspension) can bedelivered to the reagent chamber via flow channels. Additionally oralternatively reagents can be delivered to the reagent chamber by “arrayspotting” as described below.

In one aspect the invention provides a unit cell with (i) a flow channelwith contact area and (ii) a reagent chamber. In one embodiment themicrofluidic device has a plurality of unit cells, and each unit cellhas (i) a flow channel having a substrate, (ii) a membrane actuatorchamber overlying the flow channel, where the flow channel and actuatorchamber are separated by elastomeric membrane, typically a free-standingmembrane, and where an increase in pressure in the actuator chambercauses the membrane to deflect into the flow channel and contact thesubstrate at the contact area; and (iii) a reagent chamber in fluidiccommunication with the flow channel and comprising a reagent in dry formdisposed on the reagent-chamber substrate. In one embodiment at least100 unit cells of the device each contain a different reagent, differentamounts of a reagent (e.g., a dilution series, or a differentcombination of reagents).

In one aspect the invention provides a microfluidic device having aplurality of unit cells, each unit cell having a flow channel having asubstrate and detection area, an actuator chamber overlying the flowchannel, where the flow channel and actuator channel are separated byelastomeric membrane and where an increase in pressure in the actuatorchamber causes the membrane to deflect into the flow channel and contactthe substrate of the first chamber; and a reagent chamber; where eachreagent chamber is in fluidic communication with a single detectionarea; and each detection area is in fluidic communication with adetection area in two adjacent unit cell(s); a valve that can be closedto fluidically separate the detection areas and valves can be closed tofluidically separate the detection areas and reagent chambers.

Surface Patterning Using a MITOMI Device

In one aspect, the invention relates to methods for “surface patterning”a substrate of a microfluidic device by (i) masking a first portion ofthe substrate of a microfluidic chamber or channel; (ii) contactingnon-masked regions of the chamber or channel with a substrate modifyingagent; (iii) and unmasking the first portion. This results in asubstrate that has both modified and unmodified regions and may be usedto, for example, bind macromolecules to predetermined regions of thedevice substrate. In one example, a trapping element, such as amicro-mechanical membrane, makes contact with a surface and protects thecontacted surface from derivatization steps, allowing one to generate adistinctly different surface chemistry in the area where the membranecloses from the area surrounding the membrane.

Actuating the button membrane masks the region of the substrate incontact with the button membrane so that a surface modifying agentintroduced into the flow channel or chamber is excluded from the maskedregion. By using the button membrane for surface protection, complexsurface chemistries can be generated in a spatially defined area. Byvarying the design of the membrane or other movable element, surfacefeatures having a variety of shapes and sizes may be created. Usingdesigns shown in the examples, we have generated circular features witha diameter as low as 33 μm, or about ½ of spot sizes achievable bycommon quill pen based spotting (TeleChem International Inc.). Usingthis design circular features with a diameter on the order of about 5-10μm should be readily obtainable.

Using the button membrane for substrate masking can be used to generateconcentric rings of surface modification consisting of differentmolecules. This is achieved by a stepwise decrease of actuation pressurecausing the membrane to gradually disengage from the substrate, exposingpreviously unmodified regions of the protected area. These unprotectedareas may then be stepwise modified with substrate modifying molecules.

For example, Example 1 describes a chip with a substrate comprised of anepoxy glass support coated with BSA (see FIG. 2). The substrate isuniformly derivatized first with a biotinoyl-epsilon-aminocaproicacid-N-hydroxysuccinimide ester (NHS-ester biotin) to produce a biotinlayer, and second with streptavidin, which bound the biotin layer toproduce a streptaviden layer. Surface patterning was then accomplishedby closing the button membranes and flowing a biotin-tagged DNA(expression template) over the non-masked area of the device. Thebiotin-tagged DNA bound to the non-masked portion of the streptavidenlayer, producing a template layer in the non-masked areas only. Thebutton membranes were deactivated and a biotinylatedanti-penta-histidine antibody flowed over the surface. The biotinylatedantibody bound the strepavidin surface in the previously non-maskedportion of the substrate only.

Exemplary substrate modifying agents include, for example and notlimitation, proteins (e.g., antibodies, receptors, fusion proteins),nucleic acids, glycans, lipids, carbohydrates and the like. Themodifying agent may be tagged with a functional moiety such as biotin,avidin and the like. In some embodiments the modifying agent is a fusionprotein comprising an affinity tag such as His₆, maltose bindingprotein, T7 tag, and the like. The substrate modifying agent maycovalently react with the preexisting substrate or may noncovalentlyassociate with the preexisting substrate.

As illustrated in the description above, in some embodiments thesubstrate is functionalized in several steps, including multiple roundsof differential surface patterning of the substrate, to produce layers.Thus, in some cases the substrate has previously been selectivelymodified. In certain embodiments, for example, the substrate modifyingagent is biotinylated and the substrate (or a selected region thereof)has previously been modified to display an avidin. In some embodiments,substrate modifying agent comprises an antigen and the substrate (or aselected region thereof) has previously been modified to display anantibody that recognizes the antigen.

It will be understood that, as described in detail in the examples,washing and blocking steps are used as appropriate to remove unboundmaterials, block reactive moieties, etc. For example, the substratemodifying agent may be removed (e.g., by flushing the system with a washbuffer) or depleted (i.e., made unreactive or nonfunctional, e.g., byflushing the system with a compound that reacts with or modifies themodifying agent by reacting with or blocking a functional group).

Thus, in one aspect, the invention provides a method for selectivelymodifying the substrate in a unit cell of a microfluidic device by (i)masking a first portion of the substrate by contacting the first portionwith a moveable element of the unit cell; (ii) contacting non-maskedregions of the substrate with a substrate modifying agent; (iii)depleting or removing the substrate modifying agent; and (iv) unmaskingthe first portion of the substrate.

In some embodiments the substrates of only some of the unit cells of thedevice are modified or selectively modified.

Although the description above describes simultaneous actuation of allof the button membranes on the chip, the device can easily be programmedto actuate only subgroups of the button membranes, resulting in surfacepatterning of any desired complexity.

It will be recognized that, using common tagging and ligand-anti-ligandsystems the substrate can be derivatized (either uniformly or withsurface patterning) with a wide variety of molecules including, withoutlimitation, polynucleotides (e.g., DNA and RNA), polypeptides, peptides,sugars, glycans, lipids, small molecules (e.g., synthetic or naturallyoccurring organic molecules MW<1000, such as drug candidates), toxins,individual atoms, etc. Molecules can be immobilized on the existingsubstrate using any number of methods including covalent or non-covalentinteractions with the device support (e.g., glass, epoxy glass,protein-coated glass, plastic, etc.) or existing substrate, vialigand-antiligand systems (antibody-antigen, receptor-ligand,biotin-avidin, lectin-sugar, etc.) or other methods.

Mechanically Induced Trapping Of Molecular Interactions (MITOMI)

In one aspect of the invention, actuation of the button (e.g., buttonmembrane) may be used to, without limitation, physically trap surfacebound molecules in the contact area, while expelling any unbound solventand solute molecules. The trapped molecules may then be directlyobserved or tested against other compounds subsequently introduced intothe system. In addition, fluid exchanges can be carried out without lossof bound material. The trapping of bound material protects it fromsubsequent wash and fluid exchange steps allowing background moleculesto be removed and secondary reagents to be introduced into the systemwithout disturbing the trapped material. MITOMI has several advantagesover other currently available methods: it has a high intrinsicsensitivity and dynamic range, it allows the actual equilibrium bindingconstant to be observed since MITOMI literally freezes bound moleculesat equilibrium without disturbing it. Protecting the bound moleculesphysically allows for additional fluidic steps to be performed withoutloss and thus results in the highest possible downstream signal.

Molecules (including molecular complexes) may be interrogated whiletrapped using optical or other methods as described below and/or knownin the art. In one embodiment a transparent or semitransparentelastomeric material is used so that an optical signal from a trappedmolecule can be detected from outside the device.

The contacting of the membrane with the surface preferably occurs insuch a fashion that solvent and unbound solute molecules are not trappedbetween the membrane and the surface. In one embodiment initial contactof the membrane with the surface occurs medially and extends radiallyoutward. Radial closure prevents solvent pockets from forming betweenthe two interfaces and in effect creates zero dead-volume whilepreserving the equilibrium concentrations of the molecular interactionsto be detected.

One element that can be optimized or MITOMI is closing velocity. As usedherein, “closing velocity” refers to the velocity at which afree-standing membrane makes contact with the substrate. In the case ofa button membrane, radial closing velocity refers to the speed at whichthe free-standing membrane expands contact with the surface startingfrom the central contact point which extends radially outwards at theradial closing velocity. The membrane closing velocity measured as theradial closing rate should be sufficiently fast to prevent moleculesfrom dissociating while the membrane is being closed (see Example 3,infra). Generally, the velocity is sufficient that membrane whencontacting the surface efficiently traps molecules between the twointerfaces slowing down dissociational loss from the surface seenwithout membrane trapping by more than 2-3 orders of magnitude. Closingvelocity is a function of the design of the trapping element and theactuation pressure (for trapping elements actuated by an increase inpressure in an actuation chamber). Closing velocity can be determinedgenerally as described in Example 3, infra.

This method of physically trapping all surface bound molecules in thecontact area, while expelling any unbound solvent and solute moleculesallows highly sensitive secondary assays such as ELISAs to be performedon this microfluidic platform. Using this method it is possible todetect and quantify interactions between molecules, to detect specificbinding interactions, and to determine bonding affinities, among otheruses.

Subsequent to MITOMI (trapping) the trapping element can be deactuatedand a change in state of the bound molecules detected. For example, itis possible to obtain the kinetic off-rate of the interactions underinvestigation by opening the membrane and allowing the bound material todissociate while observing the rate of dissociation in real-time. Herethe rate of loss of material directly returns k_(off) of theinteraction. Since now the K_(D) and K_(off) are known, k_(on) may becomputed and all kinetic parameters are obtained for the observedinteraction. Using instrumentation capable of a simultaneously high timeresolution while interrogating several square inches or more allows oneto perform off-rate measurements in all 2400 unit cells. Likewise bytaking advantage of MSLI one can arbitrarily address a large number ofunit cells and perform off-rate assays with high time-resolution(limited only by the instrument taking the measurement) in sequencerather than in parallel.

One advantage of the present invention is that molecular interactionsthat are transient in nature and exhibit nanomolar to micromolaraffinities, leading to rapid loss of bound material or little boundmaterial in the first place can be detected and characterized.

In one aspect the invention provides a method comprising, in a unit cellof a microfluidic device, said unit cell comprising in a liquidenvironment (a) a substrate (b) a molecular complex comprising a firstmolecule immobilized in a contact area of the substrate, (c) a secondmolecule bound to the first molecule and thus indirectly bound to thesubstrate and (d) a movable element that upon actuation contacts thesubstrate in the contact area of the substrate; actuating the movableelement causing it to contact the substrate in the contact area therebyphysically trapping the first and second molecules bound to thesubstrate in the contact area while substantially expelling solvent andsolute molecules. In one embodiment the movable element is anelastomeric trapping membrane.

In a related aspect the method involves the further step of de-actuatingthe movable element (e.g., reducing pressure in a membrane actuatorchamber so that a button membrane retraces to it undeflected position).Usually the environment is changed prior to de-actuation of the movableelement (e.g., any non-bound solute is removed by doing a fluid exchangestep).

After actuating, and/or after deactuating, the movable elementmeasurements can be made of, for example, total, solid phase and/orsolution phase concentrations of the interacting molecules (e.g.,surface bound protein and bound target DNA in the case of an immobilizedDNA-binding protein interacting with a DNA molecule), and dissociationequilibrium constants or other values determined using standard methods.

Thus, in some embodiments the method further comprises detecting thecomplex of interacting molecules and/or detecting dissociation of thecomplex.

In some embodiments the effect of a third molecule on the interactionbetween the first immobilized molecule and second (bound or potentiallybound) molecule is determined. In one approach, for example, methodinvolves a) contacting the molecular complex is contacted with a thirdmolecule; b) actuating the movable element causing it to contact thesubstrate in the contact area thereby physically trapping the firstmolecule and any second molecules bound to the first moleculesubstantially expelling solvent and unbound second molecules; and c)determining the d) the effect of the third molecule on association. Inone approach, for example, method involves a) contacting the molecularcomplex with a third molecule; b) actuating the movable element causingit to contact the substrate in the contact area thereby physicallytrapping the first molecule and any second molecules bound to the firstmolecule substantially expelling solvent and unbound second molecules;c) de-actuating the movable element; and determining the d) the effectof the third molecule on dissociation of the complex. Optionally a fluidexchange step is carried out before de-actuating the movable element(i.e., the liquid environment is changed). The effect of the thirdelement on the molecular interaction is determined by comparing values(e.g., the formation of the complex of interacting molecules; thedissociation of the complex, etc.) when the third molecule is presentcompared to values when the third molecule is absent. In some variationsof this approach the effect of more than one additional molecule isdetermined (e.g., a third, fourth and fifth molecule).

It will be appreciated that information about the effect of an agent(third molecule) on an intermolecular interaction (between the first andsecond molecules) will be useful in screening for new therapeutic agents(drugs) that modulate (e.g., enhance or interfere with) intermolecularinteractions.

In some embodiments the first molecule is a polypeptide (e.g., peptide,protein, receptor, antibody, etc.), nucleic acid, carbohydrate, lipid,glycan, or small molecule. “Small molecule” is used herein to refer to acomposition, which has a molecular weight of less than about 5 kD. Smallmolecules can be nucleic acids, peptides, peptidomimetics,carbohydrates, or other organic or inorganic molecules.

In some embodiments the second molecule is a polypeptide (e.g., peptide,protein, receptor, antibody), nucleic acid, carbohydrate, lipid, glycan,or small molecule.

In some embodiments the third molecule is a polypeptide (e.g., peptide,protein, receptor, antibody), nucleic acid, carbohydrate, lipid, glycan,or small molecule.

In some embodiments, instead of adding a “third molecule” some otherperturbation of the system is studied, such as a change in temperature,introduction of kinetic energy or pressure, etc. It will also beappreciated that although for the sake of simplicity reference has beenmade to a first, second and third “molecule,” any or all of thesemolecules could actually be a molecular complex. Examples of molecularcomplexes include multi-subunit proteins or protein complexes (e.g., MHCcomplex, T cell receptor, polyketide synthase, microtubules,ribonucleoprotein complexes, proteasome, nuclear pore complex), nucleicacid complexes (e.g., triplex nucleic acids), etc.

In some embodiments, rather than measuring interactions amongstmolecules one may measure the activity rate of an enzyme of otherbiological processes. In one example, in a first step the enzyme istrapped under the membrane, followed by fluid exchange introducing asolution containing a molecular substrate for the enzyme. Opening of themembrane will allow the enzymatic reaction to commence. The resultingmodified substrate (e.g., a hydrolized substrate when the enzyme is ahydrolase) would be detected with standard art-known methods (includingthose described herein). Furthermore methods used in the detection ofkinetics of interaction discussed infra may also be applied to measuringenzymatic rates.

Due to the simplicity of the approach the method may be highlyintegrated and parallelized. To date we have performed as many as 2400distinct surface pattering reactions followed by MITOMIs on a singledevice in parallel. Furthermore an entire experiment is on the order ofa few hours in duration, which makes it faster than most other methodsof much lower degrees of parallelization. In some embodiment, the MITOMImethods described above are carried out in at least 100 unit cells of amicrofluidic device, where each of the at least 100 unit cells containsa different first molecule and/or a different second molecule and/oroptionally a different third molecule.

Detection of signal from unit cells will depend on the nature of thesignal (fluorescent, radioactive, chemiluminescent, etc.) by theassociation, disassociation, reaction, etc. Most often optical methodsare used. A variety of systems may be used including commerciallyavailable or modified microarray scanner (e.g., arrayWoRxe (AppliedPrecision), DNA microarray scanner (Axon Industries GenePix 4000B) andthe like. Furthermore surface plasmon resonance may be used fordetection of unlabeled molecular species by refractive index changes dueto mass binding to the surface of the substrate. Additionally theproduct of chemical reactions (such as catalysis, synthesis etc.) may bedetected with the above mentioned methods. When a molecular interactionis being detected typically, one or more of the molecules is labeledwith a detectable molecule (e.g., a fluorescent element). Nucleic acidscan easily be labeled during amplification or other synthesis stepsusing art-known methods. Proteins can be labeled (e.g., with afluorescent dye or macromolecule) by, for example, building a chimericprotein making use of the various GFP variants or other fluorescentproteins, introducing a modified amino acid residue (e.g., residuespecific incorporation via a modified tRNA charged with an amino acidlinked to a dye), addition of an amino reactive fluorescent dye orquantum dot in the reaction mixture, or the like.

Use Of MITOMI For Measuring Kinetic On and Off Rates

A trapping membrane may also be rapidly and repeatedly actuated anddeactuated, similar to the mechanism used for pumping using a series ofconventional monolithic valves. Rapid actuation and deactuation of thetrapping membrane can be achieved with actuation rates on the order of0.1-100 Hz, usually 0.1 to 20 Hz, often 1 to 10 Hz. Using rapidactuation of the trapping membrane can be used to control the time thedetection area is either closed, open or both. This allows one forexample to measure time dependent variables of molecular interactionssuch as the on and off rates of two or more molecules.

Measuring on Rates with MITOMI.

For illustration and not limitation, it is possible to measure theon-rate of a molecular reaction in the following way: The detection areais derivatized with Molecule A and protected by the trapping membrane ina closed state. A known concentration of Molecule B, which interactswith Molecule A, surround the detection area in solution. Opening thetrapping membrane for x amount of time (where x is 0.1 to 100 seconds)will allow Molecules B to come into contact and interact with MoleculeA. Subsequent closure of the trapping membrane will trap all Molecules Bthat were able to bind in time interval x. Consecutive repetitions ofthis approach allow one to collect y data points with time interval x(or any other time interval) and result in a curve describing the onrate of the system.

Measuring Off-Rates with MITOMI.

A similar approach can be used for measuring off-rates. In thisvariation the detection area contains Molecule A as well as Molecule Bin a complex. Repetitive opening of the trapping membrane will allowMolecule B to dissociate in x amount of time until the membrane isclosed again. Consecutive measurements of this kind will allow one tomeasure the off-rate of the system.

These approaches have advantages over real-time off-rate measurements,as many thousands of molecules can be observed at the end of each timeinterval x since the state of the system is frozen by the trappingmembrane. This allows for other detection mechanisms with limited timeresolution (but good spatial resolution and area coverage) such as DNAarray scanners to be used to quantify gain or loss of molecule B in thedetection area. Using high frequency oscillations of the button membranehas the advantage that high time-resolution measurements of on andoff-rates may be taken independently of the time-resolution of theinstrument used for taking the measurement. The time-resolution in thiscase is solely limited by the actuation frequency of the trappingmembrane.

Array Spotting

In some embodiments of the invention, reagents are introduced onto aunit cell chamber by “spotting.” Usually reagents are introduced intothe reagent chamber, but spotting may also be used to introduce reagentsinto the flow channel. By using array technology, a different reagent ordifferent combinations of reagents can be added to each unit cell. Forexample, a DNA array can be used in which each unit cell contains adifferent DNA sequence. This process involves:

(1) obtaining

-   -   (a) a solid support (e.g., an epoxy-coated glass slide), and    -   (b) a partially fabricated MITOMI device lacking a substrate;

(2) spotting one or more reagents on the solid support in a microarraypattern thereby producing a microarray of the reagents on the solidsupport; and

(3) aligning the microarray to the partially fabricated microfluidicdevice and adhering the two to produce a microfluidic device having asubstrate formed from the solid support and oriented so that each spot(or predetermined group of spots) of the array is located in a unit cellchamber of the device. Array spotting, amplification and certain otherprocesses described herein are described in copending application Ser.No. 11/698,802, “Programming Microfluidic Devices With MolecularInformation”, filed Jan. 26, 2007 (now abandoned), incorporated hereinby reference.

In one embodiment the solid support is an epoxy-functionalized glassslide, the partially fabricated microfluidic device is an elastomericdevice formed from PDMS, and bonding occurs due to an attack of theelectrophilic carbon of the epoxyde functional group by unreactedhydroxyl, alkoxyl or carboxyl groups of the PDMS. Bonding can beaccelerated by heating the device to 40° C., or can be allowed to occurat room temperature. Other substrates include, for example and not forlimitation, a tertiary layer of PDMS, unmodified glass, aldehydesurfaces, plasma treated surfaces etc.

Glass slides can be epoxy functionalized using 3-glycidoxypropyltrimethoxysilane, glycidoxypropyldimethoxymethylysilane,3-glycidoxypropyldimethyl thoxyysilane or similar molecules (e.g.,having an epoxy functional group linked to a silane group). In essence asilane molecule carrying a epoxyde functional group is either vapordeposited or absorbed in a liquid bath onto the glass surface where itthe silane moiety covalently bonds to the glass surface. Vapordeposition simply involves vaporizing the above mentioned molecule(generally at room temperature as it is a volatile) in a small chamberto which the glass slides are added. In the liquid-dip process a roughly1% solution of the above molecule in an organic solvent or mixture oforganic solvent and water is used in which the slides are dipped untilthe surface has been coated with the above mentioned molecule. Epoxycoated slides are commercially available, e.g., CEL Associates(worldwideweb.cel-1.com), Telechem International(worldwideweb.arrayit.com), Xenopore Corp. (worldwideweb.xenopore.com).

The reagents deposited in the array can be any of a wide variety ofcompounds. In various embodiments the compound is selected from thefollowing: DNA, RNA, proteins, peptides, antibodies, glycans,proteoglycans, receptors, cells, small organic molecules. Compounds thatmay be spotted include any soluble substance. Suspensions (e.g., smallcolloidal particles such as quantum dots, beads, bacterial cells andviral particles for example) can also be deposited and used to programthe device, making the spotting method extremely useful for a plethoraof applications. or small particles) that can be picked up and depositedby the arraying method used. The substrate in the area of deposition maybe derivatized to bind or otherwise interact with the spotted reagent.

A wide variety of methods are known for producing arrays on a substratesuch as a glass slide. See, for example, Heller, 2002, “DNA MicroarrayTechnology: Devices, Systems, and Applications” Ann Rev Biomed Eng4:129-53; Wingren & Borrebaeck, 2006, “Antibody microarrays: currentstatus and key technological advances” OMICS 10:411-27; Oh et al., 2006,“Surface modification for DNA and protein microarrays” OMICS 10:327-43;Uttamchandani et al., 2006, “Protein and small molecule microarrays:powerful tools for high-throughput proteomics” Mol Biosyst. 2:58-68; andUttamchandani et al., 2005, “Small molecule microarrays: recent advancesand applications” Curr Opin Chem Biol. 9:4-13, each of which isincorporated herein by reference.

Technologies for forming microarrays include both contact andnon-contact printing technologies. One example is the PixSys 5500 motioncontrol system from Cartesian Technologies (Irvine, Calif.) fitted withthe Stealth Micro-spotting printhead from TeleChem (Sunnyvale, Calif.).Contact printing technologies include mechanical devices using solidpins, split pins, tweezers, micro-spotting pins and pin and ring.Contact printing technologies are available commercially from a numberof vendors including BioRobotics (Boston, Mass.), Genetix (Christchurch,United Kingdom), Incyte (Palo Alto, Calif.), Genetic MicroSystems (SantaClara, Calif.), Affymetrix (Santa Clara, Calif.), Synteni (Fremont,Calif.), Cartesian Technologies (Irvine, Calif.) and others. Non-contactprinting technologies include “ink-jetting” type devices such as thosethat employ piezoelectrics, bubble-jets, micro-solenoid valves, syringepumps and the like. Commercial vendors of non-contact printingtechnologies include Packard Instruments (Meriden, Conn.), Agilent (PaloAlto, Calif.), Rosetta (Kirkland, Wash.), Cartesian Technologies(Irvine, Calif.), Protogene (Palo Alto, Calif.) and others. Both contactand non-contact devices can be used on either homemade or commercialdevices capable of three-dimensional movement. Motion control devicesfrom Engineering Services Incorporated (Toronto, Canada), IntelligentAutomation Systems (Cambridge, Mass.), GeneMachines (San Carlos,Calif.), Cartesian Technologies (Irvine, Calif.), Genetix (Christchurch,United Kingdom), and others would also be suitable for manufacturingmicroarrays according to the present invention.

The amount of compound required will depend on the particular nature ofthe assay, but, for proteins and nucleic acids, attomole amounts usuallyare sufficient.

The size of the microarray is typically about 1.0-2.0 cm² but may varyover a large range. The array pattern is not critical and can beoptimized for a particular device or assay. A typical spot diameter isabout 100 um (usually in the range 50-100 um, depending on the method ofspotting), with spots placed at a center-to-center spacing of about 140um (usually in the range 200-1000 um, or separating spots by at leastabout 10 um), to allow each spot to form at a distinct and separatelocation on the substrate. In one embodiment, compounds are spotted asmicroarrays with a column pitch of about of 563 μm and row pitch ofabout 281 μm.

In making an array, a small volume of a solution containing reagents isusually deposited, and the solvent allowed to evaporate leaving adesiccated reagent. The desiccated reagent spots are thus introducedinto the device and may be re-solvated by introducing liquid through theflow channel network. A carrier may be introduced to facilitatere-solvation of the reagents, for example reagents may be co-spottedwith a 1%-2% BSA solution. BSA may be added to the solution beforespotting or BSA can be co-spotted (e.g., under a spot of reagent). Theco-deposited BSA also aids in the visualization of the spots useful forthe manual alignment of the array to the microfluidic chambers. Otheradditives such as other proteins, NaCl or other salts, PEG and otherlarger organic molecules may also be used as carriers.

Since all the spots are ultimately segregated on the microfluidic deviceby specific channel geometry and active valves, it is possible to makeefficient use of arrays that are more complex than conventional arrays.Two approaches—“co-multispotting” and “neighbor spotting”—are especiallyuseful for introducing more than one solution to the same vicinity,creating complex multiplexed arrays on a MITOMI chip.

In “co-multispotting” two or more different reagent-containing solutionsare deposited on top of one another in sequential rounds of spotting, sothat several different components are located in the same place on thearray. See, e.g., FIG. 7, Panel B. This figure illustrates cospotting togenerate 3-solution combinations of 3 pairs of solutions (Pair 1=A andB; Pair 2=1 and 2 and Pair 3=alpha and beta). If any given spot containsonly one member of a pair, the total number of possible combinations is2³=8, or. In one embodiment the array is generated by spotting membersof the first pair in columns, followed by a second round of spotting ofthe same or different solutions across rows. In this example, the firsttwo rounds represent a standard two dimensional array of dimensions m×nwhere m is the number of columns and n the number of rows of the array.Printing of a three dimensional array of shape m×n×o can be accomplishedby spotting o copies of the two-dimensional array m×n. So in the caseshown in Panel B of FIG. 7 a three-dimensional array of shape m=n=o=2 isspotted on a two dimensional substrate. Likewise any array of higherdimensionality can be printed using the same technique. The deposits ofsolution A and B spotted in the same round may be spotted in sequencewithout the need of a wash step between duplicate spots. For anysubsequent round of spotting it is preferred, if pins are used fordeposit, to wash between every deposited spot due to possiblecontamination of the pin from the previously deposited spot.Co-multispotting is extremely space efficient since it requires the samearea as a standard array, and spots may be spaced with a minimal pitchmerely dictated by the pin, spotting robot and fluidic layout to whichthe array is being aligned. Co-multispotting can also be used toincrease reagent concentration per spot by multispotting the samesolution several times on the same spot, each time delivering morereagent to the amount already present on the slide.

A second approach to multiplexing by spot deposition is the method of“neighbor-multispotting” depicted in Panel C of FIG. 7. Here instead ofspotting the various solutions directly on top of one another, they arespotted immediately adjacent to one another. The total footprint of theneighboring spots is designed to fit into a single reagent chamber ofthe MITOMI device, and each group of spots is ultimately segregated onthe device. Upon resolvation the spotted reagents are allowed to mix bypassive diffusion. This approach has the disadvantage of requiring alarger footprint per spot then the co-multispotting method. However, insome applications this disadvantage is outweighed by the elimination ofcross-contamination between spotting since a pin, for example, does nottouch a previously deposited spot. Eliminating cross-contamination inthis fashion allows for significant time savings by reducing the numberof wash steps required. Like co-multispotting, neighbor multispottingcan be used to increase reagent concentration by depositing neighboringspots containing the same reagent. Neighbor multispotting typicallyresults in two or more reagents in distinct spots in an area of not morethan 1 mm², sometimes in the range of 0.1 to 0.5 mm², 0.2-0.5 mm², 100microns²-1 mm2(um²), 100 um²-200 um².

In one aspect the invention provides a method of fabricating amicrofluidic device by i) positioning an elastomeric block comprising aplurality of chamber recesses and a solid support comprising amicroarray of discrete reagent-containing regions so as to align eachreagent-containing region with a recess; ii) adhering the block to thesolid support so as to produce a plurality of chambers containingreagents. A device made from a nonelastomeric material can be alignedwith a substrate in essentially the same manner. In one embodiment themicroarray has 10 to 5,000 reagent-containing regions, more often 100 to2400 reagent-containing regions. in one embodiment each reagentcontaining region contains two or more different reagents. In oneembodiment each reagent containing region contains 1, 2 or 3 or morediscrete subregions, each containing a different reagent. In oneembodiment the microarray has a density of about 100 or more discreteregions per cm² or about 1000 or more discrete regions per cm². In oneembodiment the microarray contains 10 or more different reagents, moreoften 100 or more different reagents, and often 500 or more differentreagents.

In one aspect the invention provides a method of fabricating amicrofluidic device by i) depositing reagents on a solid support toproduce a microarray of discrete reagent-containing regions; ii)positioning an elastomeric block comprising a plurality of chamberrecesses and the reagent-containing regions so as to align eachreagent-containing region with a recess; iii) adhering the block to thesolid support so as to produce a plurality of chambers containingreagents. In one embodiment the reagents are deposited by contactprinting. In one embodiment the reagents are deposited by non-contactprinting. In one embodiment the reagents are deposited on the solidsupport robotically. In one embodiment the microarray has a density ofabout 100 or more discrete regions (corresponding to the contents of asingle chamber) per cm². In one embodiment the microarray has a densityof about 1000 or more discrete regions per cm².

In one aspect the invention provides a microfluidic device with at least100 unit cells, each unit cell having a first microfluidic chamberhaving a substrate, and a reagent in dry form disposed on areagent-containing region of the substrate where at least 100 unit cellsof the device each contains a different reagent, different amounts of areagent, or a different combination of reagents.

In Situ Transcription and Translation

As described in the Examples, in vitro transcription and/or translationcan be carried out in unit cells of the MITOMI device. The resultingproducts (nucleic acid or protein) can serve any of a variety offunctions including, for illustration and not limitation: enzymatic(e.g., catalyses a reaction in the unit cell to generate a product usedin analysis); binding (e.g., the product can act as a ligand orantiligand in an analytical or screening assay, as illustrated in theexamples); modulating (e.g., the effect of the product on a molecularinteraction between other molecules in the unit cell can be determined)and other functions.

In one embodiment, as described in the examples, in vitro transcriptionand translation (ITT) occurs when the unit cell is fludically isolatedfrom other unit cells and/or when the detection area is fluidicallyisolated from reagent chambers. ITT can be used to produce analytes orreactants in a unit cell chamber or flow channel.

In one embodiment the expression template (the nucleic acid from whichtranscripts are made) is immobilized. Preparation and use of expressionvectors and linear expression templates is well known. In one embodimentthe expression template is produced using a two-step amplification asdescribed in copending application No. 60/762,344, incorporated hereinby reference. In one embodiment the template is immobilized on the unitcell substrate (e.g., optionally using a tag such as biotin) and a wheatgerm extract (or other transcription/translation system) is addedsubsequently. In another embodiment, the transcription/translationsystem is added together with the template and distributed to individualunit cells, which are then sealed and protein synthesis allowed toproceed. For example, in one embodiment, after surface patterning thedevice is loaded with wheat germ based ITT mixture containing linear DNAtemplate coding for a protein to be synthesized and each unit cell isisolated by closing a set of micromechanical valves. The device isincubated at 30° C. for 90 min to complete protein synthesis.

In one embodiment, multiple different proteins are produced in situ inthe same unit cell. These multiple proteins may be variants of a basesequence, may interact with each other or compete for interaction withanother molecule, may function in a common synthetic or metabolicpathway, may have another relationship or may be unrelated.

Exemplary Analysis

For illustration a hypothetical analysis is presented in this section.It should be understood that this description is simply to illustratecertain aspects of the invention and is not intended to limit theinvention in any way.

Step 1. Prepare linear expression template (“target DNA”) of geneencoding a ligand (“target protein”). 2. Spot plain epoxy support with2400 unique spots of a peptide library, in which at least some of thepeptides are believed to be an anti-ligand for the target protein, toform a microarray. The peptides are tagged with a fluorophore. 3. Alignand adhere an elastomeric block comprising a plurality of chamberrecesses so as to align each reagent-containing region with a recesscorresponding to a “reagent chamber,” thereby producing a microfluidicdevice containing unit cells having (i) a reagent chamber in whichtarget peptide is deposited on the substrate; (ii) a flow channel with adetection area in fluidic communication with the reagent chamber andwith detection areas of two adjacent unit cells; (iii) valves betweenthe detection areas and between the detection area and the reagentchamber (allowing the reagent chamber and detection area to be isolatedby closing the valves); and a membrane actuator chamber positioned overthe detection area and separated from it by a thin deflectableelastomeric membrane. 4. Fluidically isolate the reagent chamber byclosing the appropriate valve. 5. Differentially derivatize thesubstrate of the detection area, so that the detection area, and noother area of the substrate, has an stepavidin substrate. 6. Introduce abiotinylated penta-histidine tagged antibody against the target protein,which is immobilized in the detection area. 7. Introduce ITT containingthe target DNA (linear template) into the detection area and closevalves to isolate chamber. The target protein is synthesized and thenimmobilized by binding to antibody. 8. Open valve between detection areaand reagent chamber; allow peptides to solvate and bind the immobilizedtarget protein. 9. Actuate the button membrane to mechanically trap anypeptide bound to immobilized target protein molecular interactionstaking place on the surface allowing all solution phase molecules to bewashed away without loss of surface bound material. 10. Fluorescent scanof the device to detect quantities of trapped molecules, in solutionmolecules. 11. Remove any unbound (solution phase) peptides by flushingchamber with buffer. 12. De-actuate button membrane and detect boundpeptide (showing interaction) 13. Measure rate of dissociation ofpeptide from target protein.

EXAMPLES Example 1 Surface Patterning and MITOMI

This example describes a prototypical MITOMI device. A device wasfabricated, a protein-based surface chemistry for protein binding wasgenerated, and protein capture and MITOMI were carried out.

Additionally the effect of varying chamber diameters on the effectivesurface contacting area was also studied.

Experimental Methods

A) Flow and Control Mold Manufacturing

Two 3-inch silicone wafers were treated with hydroxy-methyl silane(HMDS, Sigma) for 2 minutes followed by spin coating with the positivetone photoresist SPR 220-7.0 (Rohm & Haas) in two consecutive spinsteps. The first spin step was performed at 500 rpm for 5 secondsfollowed by 3000 rpm for 60 seconds. The wafers were then soft-baked at105° C. for 90 seconds. The flow and control patterns were transferredto the molds using two separate transparencies printed with theirrespective patterns at 20,000 dpi (CadArt). Exposure was done at 365 nmon a MA-6 Mask aligner (Karl Suss) for 15 seconds. Subsequently themolds were developed in a 5:1 bath of distilled water and 2401 developer(Shipley) respectively until the channel patterns were visible and anyexcess background removed.

B) Chip Fabrication

For the control layer 36 grams of a 5:1 ratio of Part A to Part BSylgard (DowCorning) were mixed together in a Hybrid mixer (ThinkyCorporation) for 1 minute followed by degassing for 2 minutes. Themixture was then poured on top of the control layer mold sitting in aPetri dish lined with aluminum foil and further degassed in a vacuumchamber for roughly 5 minutes. The flow layer consisted of 31.5 grams ofa 20:1 mixture of part A to part B Sylgard, which was also mixed for 1minute and degassed for 2 minutes in the hybrid mixer. The flow layermixture was then spun onto the flow layer mold at 3000 rpm for 30seconds with a 15 second up-ramp and no down-ramp. Both layers wherecured at 80° C. for 30 minutes after which the control layer was cut andremoved from the control layer mold. Access holes were punched with a 21gauge manual puncher. The control layer was then manually aligned to theflow layer and the two where bonded together at 80° C. for 2-3 hours.Finally the monolithic device was peeled of the flow layer mold andaccess holes to the flow layer were punched with the same puncher asbefore.

The finished chip was placed on an epoxy slide (CEL Associates)previously coated with bovine serum albumin (BSA, Sigma) by submergingthe slide in a 2% BSA phosphate buffered saline (PBS) solution for 2hours followed by a dH2O wash and dying under a stream of Nitrogen.

C) Preparation of Control Lines and Protection of Surface

All the control lines of the device were connected to tubes filled withdistilled water and actuated at 5 psi until all control channels weredead-end filled with liquid. During the experiment, control lines whereactuated at a pressure of 15 psi.

D) Activation of BSA Surface Layer

Initial activation of the BSA surface layer was performed by introducing4.4 mM Biotinoyl-epsilon-aminocaproic acid-N-hydroxysuccinimide ester(NHS-ester biotin, Roche) in a 10% solution of dimethyl formamide (DMF)in PBS at a constant flow rate at 5 psi for 48 minutes. This wasfollowed by a 10 minute long wash with PBS. The surface was then coatedwith streptavidin (Roche) by flowing a 500 μg/mL streptavidin (Roche)PBS solution into the device for 23 minutes, followed by a 5 minute PBSwash.

The button membranes were closed and washing continued for an additional5 minutes. Once washing was complete the non-protected area of thedevice was derivatized with a PCR derived linear expression templatecoding for MAX isoform A N-6×His which was flowed through the serpentinechannel for 55 minutes. The expression template was covalently coupledon the 5′ end to a Cy3 fluorophore and on the 3′ end to biotin. Anyunbound or loosely bound template was removed in a 8 minute long PBSwash step.

Once the channels were cleared of residual templates, a 1:1 solution ofbiotinylated anti-penta-histidine antibody (Qiagen) in 2% BSA PBS wasintroduced into the serpentine for 5 minutes before de-actuating thebutton membrane and allowing the antibody to coat the now accessiblestreptavidin surface for 10 minutes under constant flow. This was againfollowed by a 8 minute long PBS wash.

In order to synthesize protein in situ using the previously depositedlinear DNA, a standard wheat germ based in vitrotranscription/translation reaction (Promega) spiked with 1 μLtRNALys-bodipy-fl and 1 μL of a 1/10 dilution of a dsDNA oligomercontaining a transcription factor binding sequence, was introduced intothe serpentine for 9 minutes. Each unit cell was then segregated and theentire device was incubated for roughly 2 hours on a 31.9° C. warmhotplate.

The button membrane was then closed one more time trapping any surfacebound protein-DNA complexes and the serpentine flushed with PBS for 19minutes.

FIG. 2 shows a schematic of the surface chemistry that was generated onthe device as well as the process of protein synthesis, capture andMITOMI. Colored boxes indicated fluorescently labeled molecules,green=fluorescein, yellow=Cy3 and red=Cy5. Panel A shows the finalsurface chemistry just prior to introduction of the in vitrotranscription/translation reagents. Each grey block represents amonolayer consisting of the indicated molecule. Panel B describes theprocess of protein synthesis using the deposited linear expressiontemplates. The synthesized MAX isoA protein diffuses to the antibodycoated surface and is pulled down via its N-terminal 6×Histidine tag.The free Ebox DNA molecules, introduced with the ITT mix, are recognizedby MAX iso A and likewise pulled down to the surface. In Panel C MITOMIis performed by closure of the button membrane, trapping any boundmaterial and expunging any unbound material. This schematic correspondsto the image in FIG. 3, panel B. Panel D shows the final state of thedevice after the last PBS wash removing any unbound material from theadjacent material. This schematic corresponds to the image in FIG. 3,Panel B.

Results

FIG. 3 summarizes the results of the experiment. Panel A shows anAutoCad schematic of a subsection of the device used in this experiment.Flow layers are outlined by blue lines and the control layer situated ontop of the flow layer is outlined in red. Note the red circles ofvarying diameter. These constitute the chambers creating the buttonmembrane. In Panel B a section of a scan taken with a modified DNA arrayscanner (ArrayWoRxE, Applied Precision) is shown. The image was acquiredin the Cy5 band, and thus shows the relative fluorescent intensity ofthe Ebox DNA that was spiked into the ITT reaction. The intensity scales(in grey scale or in false colors from green to red, with green beingthe lowest concentration and red the highest.) It can be seen that onlythe flow channel but not the chamber show signal, which is to beexpected since the chambers were not filled and remained empty.Furthermore it is apparent that each unit cell contains a concentricring of low intensity of varying diameter, with a high intensitybull's-eye. This pattern stems from the button membrane closure andMITOMI and is indicative of two things. First the bright bull's-eyeoriginates exclusively from surface bound Ebox-DNA specifically bound byMAX iso A. Secondly, the surrounding low intensity ring indicates thatno non-specific trapping of molecules occurs. The low intensity ring isseen because the button membrane was closed at a higher pressure thanused originally to protect the surface from derivatization with linearexpression template. Meaning that the membrane now contacts a largersurface area, but Ebox-DNA is only trapped where it was pulled down byMAX isoA and not where it can only make non-specific interactions withthe surface bound linear-expression template. Panel C shows the samearea of the device after a PBS wash step indicating that fluid exchangemay be performed without loss of trapped material. It should be notedthat the MAX iso A-Ebox DNA interaction has a high intrinsic k_(off)rate on the order of 0.2 sec-1 and a overall affinity in the lownanomolar range. The extent of non-specific trapping of solute materialby closure of the button membrane is described in further detail inExample 2, infra. In order to assess how contact diameter depends onchamber size we designed a device that contains chambers of varyingsizes ranging from 180 μm down to 80 μm in 20 μm steps. Initialactuation was tested at varying pressures to understand what minimalpressure is required to put each of the membranes into contact with thesurface. The results are summarized in Table 1 and show that all testedmembranes may be deflected and closed and that the actuation pressuresfall within reasonable bounds of up to 20 psi. Additionally thedependence on the membrane-surface contact area as a function of chamberdiameter at a constant closing pressure of 15 psi was also determined byplotting the resulting spot diameter as a function of chamber diameter(FIG. 4). The results show hat the resulting contact surface is directlyand linearly dependent on chamber diameter with a slope close to 1.Therefore the actual area to be contacted can easily be predicted andmodulated by increasing the chamber diameter, closing pressure as wellas flow channel height. Where the latter two parameters will most likelyonly affect the intercept of the line and not the slope.

TABLE 1 Spot size dependence and uniformity Membrane Initial closingSpot diameter Std. dev. diameter (μm) pressure (psi) at 15 psi (μm) (μm)180 6.5 102 5 160 6.5 80 5 140 6.5 54 5 120 7.5 33 4 100 19 — — 80 19 ——

Example 2 Establishing Detection Sensitivity Levels

In order to address the question what the lower detection bounds are forthis system we determined at what concentrations non-specific trappingoccurs. To do so a device similar to the one described in Example 1 wasused. We tested three different Ebox-DNA sequences: Ebox, Er and Enotcontaining the following E-box binding sequences respectively: CACGTG,CAGGTG and TGATGC. MAX iso A binds the Ebox sequence with a strength inthe nanomolar range with decreasing affinities for Er and Enot. Enotbinds only non-specifically via interactions with the phosphate backboneand its' affinity is considered negligible falling into the μM regimeand considered non-specific. These three sequences when tested in oursystem return the approximate affinites of 166 nM, 1.3 μM and 23 μM forEbox, Er and Enot respectively when bound to the surface by aC-terminally 6×Histidine tagged MAX iso A version (FIG. 5). Moreimportantly when MAX iso A without an affinity tag is used all measuredaffinities drop to roughly 50 μM

In this experiment 50 μM was the value where non-specific bindingdominates in this system. It is noteworthy that using MITOMI it ispossible to resolve binding differences between non-specific binding dueto DNA phosphate backbone interactions as was the case with Enot bindingto MAX iso A C-His, which using standard methods would have been lost inthe overall non-specific binding observed with tag-less versions ofMAXiso A. Affinities in biological systems can be as low as a few dozenμM, which we are still able to capture in our system. Any affiniteslower then the ones reported herein can be considered non-specific andthus likely not important in biological processes. Our system also hasbeen shown to have a dynamic range of at least two orders of magnitude,ranging from about 50 μM to roughly 200 μM. Higher affinities shouldalso be detectable, since they are generally easier to detect, extendingthe dynamic range by at least another order or two.

In summary we have developed a method for the highly-parallel andsensitive detection of molecular interactions that may be applied to abroad range of interactions and does not require extensive opticalsetups for detection. Detection sensitivity and dynamic range cover allbiologically relevant affinities making MITOMI broadly applicable forthe detection of molecular interactions.

Example 3 A Microfluidic Platform to Measure Low Affinity InteractionsEnables Comprehensive Characterization of Transcription Factor BindingEnergy Landscapes and Prediction of Gene Regulation

This example describes the use of MITOMI to map the binding energylandscapes of four eukaryotic transcription factors (TFs) belonging tothe basic helix-loop-helix (bHLH) family by collecting over 41,000individual data points from more than 17 devices and covering titrationsover 464 target DNA sequences. These binding energy topographies allowedus to 1) predict in vivo function for two yeast TFs, 2) make acomprehensive test of the base additivity assumption, and 3) test thehypothesis that the basic region alone determines binding specificity ofbHLH TFs. bHLH motifs represent the third largest TF family ineukaryotes and regulate a wide variety of cellular functions rangingfrom cell proliferation and development to metabolism. Information inthis Example is found in Maerkl S J and Quake S R, 2007, “A systemsapproach to measuring the binding energy landscapes of transcriptionfactors” Science 315:233-7, incorporated herein by reference.

We studied isoforms A and B of the human TF MAX, which together withother bHLH members play a role in cellular proliferation and manycancers. We also studied the yeast TFs Pho4p and Cbf1p; the formerregulates phosphate metabolism, while the latter regulates methioninesynthesis as well as chromosome segregation, serving a structural rolein the kinetochore. bHLH TFs generally bind to a consensus sequence of5′-CANNTG-3′ called “enhancer box” (E-box), which was later found to bethe second most conserved motif in higher eukaryotes. Members of thebHLH family show mid to low nanomolar DNA binding affinities and haveoff-rates above 10⁻² s⁻¹ for their consensus sequences with orders ofmagnitude higher off-rates for non consensus sequences. This transiencemakes the use of conventional microarrays impractical.

Overview

The TF binding energy topographies were measured with highly integratedmicrofluidic (MITOMI) devices (Thorsen et al., 2002, Science 298:580)containing 2400 independent unit cell experiments (FIGS. 6A and 6B).Each device is controlled by 7,233 valves fabricated by multilayer softlithography (MSL) (Unger et al., 2000, Science. 288:113) and programmedwith a 2,400 spot DNA microarray (Shena et al., 1995, Science 270:467).The 2,400 chambers are arranged into 24 rows addressed via a resistanceequalizer (FIG. 6A); this ensures that flow velocities are equal acrossall rows, resulting in uniform surface derivatization and TF deposition.(Also see WO 2006/071470 describing a distribution manifold.)

The device was designed in AutoCAD2004 (Autodesk, Inc.) and each layerreproduced as a chrome mask at 20,000 dpi (Fineline-Imaging). Flow moldswere fabricated on 3″ silicon wafers (Silicon Quest International)coated with hexamethyldisilazane (HMDS) in a vapour bath for 2 min. Thewafers were then spin coated with SPR 220-7 (Shipley) initially at 500rpm for 5 s followed by 4000 rpm for 60 s yielding a substrate height ofaround 6-7 μm. The molds were baked at 105° C. for 90 s followed by a 15s I-line exposure on a MA6 contact mask aligner (Karl Suss). Next themolds were developed with 1:5 2401 developer (Microposit) in dH2O.Finally the molds were annealed at 120° C. for 20 min. Control moldswere fabricated on 3″ silicon wafers by spin coating SU-8 2025(MicroChem) at 2700 rpm for 80 s followed by a 65° C. bake for 2 min,95° C. for 5 min and a final step of 65° C. for 2 min. The wafers werethen exposed for 10 s on the I-line, followed by a post-exposure bakeseries of 65° C. for 2 min, 95° C. for 12 min and 65° C. for 2 min. Thewafers were then developed in SU-8 developer for 90 s followed by anacetone and isopropanol wash. One wafer from each control and flow waferset was selected and used for all subsequent microfluidic devicefabrication. The microfluidic devices were fabricated essentially asdescribed previously (Thorsen et al., 2002, Science 298:580).

a) Target DNA Synthesis

We synthesized libraries of Cy5 labeled target DNA sequences whichcomprehensively cover the E-box motif and flanking bases by permuting upto four bases at a time.

All small dsDNA oligos serving as targets for transcription factorbinding were synthesized by isothermal primer extension in a reactioncontaining 6.7 μM 5′CompCy5, 10 μM library primer, 1 mM of each dNTP, 5units Klenow fragment (3′→5′ exo-), 1 mM dithiothreitol 50 mM NaCl, 10mM MgCl2 and 10 mM Tris-HCl, pH7.9 in a final volume of 30 μL. Allreactions were incubated at 37° C. for 1 h followed by 20 min at 72° C.and a final annealing gradient down to 30° C. at a rate of 0.1.0 sec-1.We added 70 μL of a 0.5% BSA dH2O solution to each reaction andtransferred the entire volume to a 384 well plate in which a 6 folddilution series was established with final concentrations of 5′CompCy5of 2 μM, 600 nM, 180 nM, 54 nM, 16 nM and 5 nM.

b) DNA Arraying and Device Alignment

Dilution series for each target DNA sequence were spotted as microarrayswith a column and row pitch of 563 μm and 281 μm, respectively. Thesearrays were used to program the microfluidic devices by aligning eachspot to a unit cell.

Programming devices with microarrays simplifies the microfluidicinfrastructure and increases unit cell density. The use of microarraysfor device programming is highly modular as any soluble substance orsuspension may be arrayed and it provides an elegant and efficientsolution to the world to chip interface problem. Approximately attomolesof DNA and TF are required for each data point.

All target sequences were spotted with an OmniGrid Micro (GeneMachines)microarrayer using a CMP3B pin (TeleChem International, Inc.) fordelivery onto epoxy coated glass substrates (CEL Associates). Eachsample solution contained 1% BSA in dH2O to prevent covalent linkage ofthe target DNA to the epoxy functional groups as well as forvisualization during alignment. After spotting the arrays were qualitycontrolled on a GenePix4000b (Molecular Devices). The arrays could thenbe stored in the dark at room temperature until aligned to amicrofluidic device. Device alignment was done by hand on a SMZ1500(Nikon) stereoscope and bonded overnight in the dark on a heated plateat 40° C.

c) Linear Template Synthesis

To avoid time consuming cloning and protein synthesis/purificationsteps, the TFs are synthesized in situ via wheat germ based in vitrotranscription/translation (ITT). We designed a two-step PCR method thatgenerates linear expression ready templates directly from yeast genomicDNA or cDNA clones. This approach allowed us to not only rapidly screennew TFs, but also to easily create and test structural chimeras.

Linear expression templates were generated by a two step PCR method(FIG. 9) in which the first step amplifies the target sequence and thesecond step adds required 5′UTR and 3′UTR for efficient ITT. Pho4 N orC-His tagged and Cbf1 N or C-His tagged versions were amplified 1 fromyeast genomic DNA as follows: The first step PCR reaction contained 1 μMof each gene specific primer, 10 ng μL-1 yeast genomic DNA (SeeGene),200 μM of each dNTP and 2.5 units of TAQ enzyme mixture (Expand HighFidelity PCR system, Roche) in a final volume of 50 μL. The reaction wascycled for 4 min at 94° C., followed by 30 cycles of 30 s at 94° C., 60s at 53° C. and 90 s at 72° C. followed by a final extension of 7 min at72° C. The products were then purified on spin columns (QIAquickPCR,Qiagen) and eluted in 75 μL of 10 mM TrisCl, pH 8.5. The purifiedproduct then served as template in the second PCR reaction using 2 μLfirst PCR product, 5 nM 5′ext1 primer, 5 nM 3′ext2 primer, 200 μM ofeach dNTP and 2.5 units of TAQ enzyme mixture (Expand High Fidelity PCRsystem, Roche) in a final volume of 100 μL. The reaction was cycled for4 min at 94° C. followed by 10 cycles of 30 s at 94° C., 60 s at 53° C.and 90 s at 72° C. followed by a final extension of 72° C. for 7 min.After this first round of extension 2 μL of 5 μM 5′final Cy5 and 5 μM3′final in dH2O were added to each reaction and cycling was continuedimmediately at 94° C. for 4 min followed by 30 cycles of 30 sec at 94°C., 60 sec at 50° C. and 90 s at 72° C. followed by a final extension of72° C. for 7 min. The final product was then purified on spin columnsand eluted in 100 μL 10 mM TrisCl, pH8.5 or used directly in ITTreactions. Linear expression templates for MAX iso A, MAX iso B weresynthesized essentially as above except that bacterial cDNA clones (MGC)lysed in 2.5 μL Lyse n′ Go buffer (Pierce) at 95° C. for 7 min whereused as template in an Expand High Fidelity PCR reaction (Roche). Thefirst PCR product was purified using the Qiaquick 96 PCR purificationkit (Qiagen) and eluted in 80 μL of 10 mM TrisCl, pH 8.5. To assess thefidelity of these multi-step PCR reactions and to ascertain that nopoint mutations accumulated during the reaction we submitted finalproducts of MAX iso B notag, MAX iso B C-His, PHO4 C-His and CBF1 N-Histo sequencing (Biotech Core). The resulting sequences showed extremelyhigh-fidelity with no accumulation of point mutations (data not shown).

d) Derivatization

During surface derivatization, a circular area is masked with the buttonwhile the rest of the surface is passivated with biotinylated bovineserum albumin. When the button is released, the previously protectedcircular area is specifically derivatized with biotinylated anti-His5antibody (FIG. 8C-G). After surface patterning the device is loaded withwheat germ based ITT mixture containing linear DNA 5 template coding forthe TF to be synthesized and each unit cell is isolated by closing a setof micromechanical valves. The device is incubated at 30° C. for 90 minto complete TF synthesis, solvation of target DNA, and equilibration ofTF and target DNA (FIGS. 8H-I).

For the initial surface derivatization steps the chamber valves remainedclosed to prevent liquid from entering the chambers containing thespotted DNA targets (FIG. 8C). First, all accessible surface area wasderivatized by flowing a solution of biotinylated BSA (Pierce)resuspended to 2 mg/mL in dH2O for 30 min through all channels, followedby a 10 min PBS wash (FIG. 8D). Next a 500 μg/mL Neutravidin (Pierce)solution in PBS was flown for 20 min, followed by a 10 min PBS wash(FIG. 8E). Next, the “button” membrane was closed and the PBS washcontinued for an additional 5 min. Then all remaining accessible surfacearea was passivated with the same biotinylated solution as above for 30min, followed by a 10 min PBS wash (FIG. 8F). Finally a 1:1 solution ofbiotinylated-penta-histidine antibody (Qiagen) in 2% BSA in PBS wasloaded for 2-5 min, after which the “button” membrane was opened andflow continued for 20 min, again followed by a 10 min PBS completing thesurface derivatization procedure (FIG. 8G).

e) In Vitro Transcription/Translation (ITT)

Following derivatization a standard 25 μL TNT T7 coupled wheat germextract mixture (Promega) was prepared and spiked with 1 μLtRNALys-bodipy-fl (Promega) and 2 μL of linear expression ready templatecoding for the appropriate transcription factor. The mixture wasimmediately loaded onto the device and flushed for 5 min, after whichthe chamber valves were opened allowing for dead end filling of thechambers with wheat germ extract (FIG. 8H). The chamber valves wereagain closed and flushing continued for an additional 5 min. Next thesegregation valves separating each unit cell were closed followed byopening of the chamber valves allowing for equilibration of the unitcell by diffusion. The entire device was heated to 30° C. on atemperature controlled microscope stage and incubated for up to 90 min(FIG. 8I). After the incubation period the device was imaged on amodified arrayWoRxe (AppliedPrecision) microarray scanner.

f) MITOMI

Next we performed MITOMI by closing the “button” membrane (FIG. 8J) aswell as the chamber valves (FIG. 8K) followed by a 5 min PBS wash (FIG.8L) after which the device was imaged once more to detect the trappedmolecules. MITOMI characterization To measure the effect of buttonclosing rate on DNA trapping we measured the button closing rates of 640chamber and 2400 chamber devices. The buttons were closed at variouspressures ranging from 12 psi to 24 psi in 3 psi steps to modulatebutton closing velocities. Movies were taken of the button closing atthese various pressures using a digital camera (DFW-V500, Sony) at 25fps. The radial button closing velocities were extracted from thesevideos for both devices at all pressures. Closing velocities differbetween the two devices due to slight differences in architecture of thebutton, where the 640 chamber device had a narrower channel connectingthe button with the feeding channel. In order to measure the effect ofclosing velocity on DNA trapping efficiency we measured the resultingratio of trapped DNA to protein under the button after closing atvarious velocities. We performed all measurements at closing pressuresof 15 psi-18 psi reaching velocities of 4.6 μm/sec and above and thusare in a region were the closing velocity is sufficiently fast and noDNA loss is observed. In order to assess the effectiveness of themechanical trapping of DNA by the PDMS membrane we measured how much DNAis lost while the button is in a closed state. All experiments wereperformed on a 640 chamber version of the original device with a TNNNGTGlibrary and MAX iso A C-His as the transcription factor. Two experimentswere performed with various measurement intervals. The first experimentconsisted of measurement intervals of 30 min for two hours followed by afinal long term measurement 15 hours into the experiment. On a seconddevice the measurement interval was extended to 60 min for four hoursfollowed by a final 5 measurement at 21 hours. We then fit exponentialfunctions to all four time-series and plotted the resulting rateconstants (not shown). In order to separate the contribution ofbleaching to the actual mass loss rate we plotted the measured rateconstants as a function of the measurement interval and fit a linearregression, of which the intercept represents the actual mass loss ratewith a value of 0.0009 sec⁻¹ (not shown). We therefore observe a smallmass loss of DNA from beneath the button on very long time scales, mostlikely due to lateral diffusion, but the loss is negligible over thetime course of a normal experiment.

To ascertain the reproducibility of MITOMI we compared experiments fromdifferent days and devices for all four TFs studied (not shown). Allcomparisons show good correlation of values including the low affinityregime. The fact that low affinity measurements correlate indicates thatthey do not lie near the detection limit, determined to be around 18 μM(data not shown). To arrive at a global measurement error that includesboth biological as well as technical noise we compared all N andC-terminally tagged TF datasets, yielding a global measurement error of19% (not shown). Additional negative controls such as a no protein aswell as a no-epitope tag MAX A control showed no non-specific trappingof DNA (data not shown).

Image and Data Analysis

All images were analyzed with GenePix3.0 (Molecular Devices). For eachexperiment two images where analyzed. The first image taken after the60-90 min incubation period, was used to determine the concentration ofsolution phase or total target DNA concentration (Cy5 channel). Thesecond image taken after MITOMI and the final PBS wash was used todetermine the concentration of surface bound protein (FITC channel) aswell as surface bound target DNA (Cy5 channel). Dissociation equilibriumconstants were determined for each experiment using Prism 4 (GraphpadSoftware) by performing global nonlinear regression fits using a one 6site binding model to the data plotted as surface bound target DNA (RFU)divided by surface protein concentration (RFU) (or effectivelyfractional occupancy) as a function of total target DNA concentration(RFU). The Bmax parameter was set equal to the plateau of the consensussequence and used for all linefits. These relative Kds (RFU-1) were thentransformed into absolute Kds (M-1) using a calibration curve previouslyestablished by measuring known quantities of 5′CompCy5 primer (data notshown). ΔΔGs were calculated with ΔΔG=RT*ln(Kd/Kd_(ref)) at atemperature of 298 K. The highest affinity sequence was always chosen asthe reference. We estimated our measurement error by plotting affinitiesmeasured of all N-terminally tagged transcription factors versus theirrespective C-terminal variants. We adjusted all slopes of the linearregression fits to be uniform for all transcription factors. Ourobserved variance was heteroscedastic. We therefore applied a lntransform to our data which resulted in constant variance from which weobtained σ values of 0.17 and 0.40 for one and two σ respectively.Re-transforming these values yields ^σ=0.19x and 2^σ=0.49x or 19% and49% respectively.

In Silico Model

Our measurements agree with previous reports that the optimal bindingsequence for all four TFs is CACGTG for N-3-N3. We measured consensusbinding affinities of 67.0 nM, 73.1 nM, 11.1 nM and 16.6 nM for MAXisoform A, isoform B, Pho4p and Cbf1p, respectively. The bindingaffinity of MAX to a slightly different sequence has been measuredindependently and is in agreement with our results for that sequence(Park et al., 2004, Biochim. Biophys. Acta 1670:217). Each bindingenergy landscape exhibits topographic fine structures such as affinityspikes for sequences with a one base spacer between the two half-sites(CACGGTG for example) as well as consensus neighbors CATGTG, CTCGTG andCAGGTG. These fine structures often lie in the low affinity regime (withoff-rates on the order of 2-20 s⁻¹) and have thus far not been observedwith other methods. The binding energy landscapes for both MAX isoformsare more rugged than the landscapes of Pho4p and Cbf1p, showing strongaffinities for consensus neighbors, whereas Pho4p and Cbf1p aresingularly specific for the E-box consensus. These differences intopography are intriguing since crystal structures of truncated versionsof MAX and Pho4p show 6 that both TFs make essentially the same basespecific contacts. Therefore similar base specific contacts give rise torecognition of the same consensus sequence but not necessarily tosimilar overall binding topographies.

GENERAL MATERIALS AND FABRICATION METHODS

The methods used in fabrication of a microfluidic device will vary withthe materials used, and include soft lithography methods, microassembly,bulk micromachining methods, surface micro-machining methods, standardlithographic methods, wet etching, reactive ion etching, plasma etching,stereolithography and laser chemical three-dimensional writing methods,modular assembly methods, replica molding methods, injection moldingmethods, hot molding methods, laser ablation methods, combinations ofmethods, and other methods known in the art or developed in the future.A variety of exemplary fabrication methods are described in Fiorini andChiu, 2005, “Disposable microfluidic devices: fabrication, function, andapplication” Biotechniques 38:429-46; Beebe et al., 2000, “Microfluidictectonics: a comprehensive construction platform for microfluidicsystems.” Proc. Natl. Acad. Sci. USA 97:13488-13493; Rossier et al.,2002, “Plasma etched polymer microelectrochemical systems” Lab Chip2:145-150; Becker et al., 2002, “Polymer microfluidic devices” Talanta56:267-287; Becker et al., 2000, “Polymer microfabrication methods formicrofluidic analytical applications” Electrophoresis 21:12-26; U.S.Pat. No. 6,767,706 B2, e.g., Section 6.8 “Microfabrication of a SiliconDevice”; Terry et al., 1979, A Gas Chromatography Air AnalyzerFabricated on a Silicon Wafer, IEEE Trans, on Electron Devices, v.ED-26, pp. 1880-1886; Berg et al., 1994, Micro Total Analysis Systems,New York, Kluwer; Webster et al., 1996, Monolithic Capillary GelElectrophoresis Stage with On-Chip Detector in International ConferenceOn Micro Electromechanical Systems, MEMS 96, pp. 491496; and Mastrangeloet al., 1989, Vacuum-Sealed Silicon Micromachined Incandescent LightSource, in Intl. Electron Devices Meeting, IDEM 89, pp. 503-506.

In preferred embodiments, the device is fabricated using elastomericmaterials. Fabrication methods using elastomeric materials will only bebriefly described here, because elastomeric materials, methods offabrication of devices made using such materials, and methods for designof devices and their components have been described in detail (see,e.g., Thorsen et al., 2001, “Dynamic pattern formation in avesicle-generating microfluidic device” Phys Rev Lett 86:4163-6; Ungeret al., 2000, “Monolithic microfabricated valves and pumps by multilayersoft lithography” Science 288:113-16; Linger et al., 2000, Science288:113-16; U.S. Pat. No. 6,960,437 (Nucleic acid amplificationutilizing microfluidic devices); U.S. Pat. No. 6,899,137(Microfabricated elastomeric valve and pump systems); U.S. Pat. No.6,767,706 (Integrated active flux microfluidic devices and methods);U.S. Pat. No. 6,752,922 (Microfluidic chromatography); U.S. Pat. No.6,408,878 (Microfabricated elastomeric valve and pump systems); U.S.Pat. No. 6,645,432 (Microfluidic systems including three-dimensionallyarrayed channel networks); U.S. Patent Application publication Nos.2004/0115838, 20050072946; 20050000900; 20020127736; 20020109114;20040115838; 20030138829; 20020164816; 20020127736; and 20020109114; PCTpatent publications WO 2005/084191; WO05030822A2; and WO 01/01025; Quake& Scherer, 2000, “From micro to nanofabrication with soft materials”Science 290: 1536-40; Xia et al., 1998, “Soft lithography” AngewandteChemie-International Edition 37:551-575; Unger et al., 2000, “Monolithicmicrofabricated valves and pumps by multilayer soft lithography” Science288:113-116; Thorsen et al., 2002, “Microfluidic large-scaleintegration” Science 298:580-584; Chou et al., 2000, “MicrofabricatedRotary Pump” Biomedical Microdevices 3:323-330; Liu et al., 2003,“Solving the “world-to-chip” interface problem with a microfluidicmatrix” Analytical Chemistry 75, 4718-23,” Hong et al, 2004, “Ananoliter-scale nucleic acid processor with parallel architecture”Nature Biotechnology 22:435-39; Fiorini and Chiu, 2005, “Disposablemicrofluidic devices: fabrication, function, and application”Biotechniques 38:429-46; Beebe et al., 2000, “Microfluidic tectonics: acomprehensive construction platform for microfluidic systems.” Proc.Natl. Acad. Sd. USA 97:13488-13493; Rolland et al., 2004,“Solvent-resistant photocurable “liquid Teflon” for microfluidic devicefabrication” J. Amer. Chem. Soc. 126:2322-2323; Rossier et al., 2002,“Plasma etched polymer microelectrochemical systems” Lab Chip 2:145-150;Becker et al., 2002, “Polymer microfluidic devices” Talanta 56:267-287;Becker et al., 2000, “Polymer microfabrication methods for microfluidicanalytical applications” Electrophoresis 21:12-26; Terry et al., 1979, AGas Chromatography Air Analyzer Fabricated on a Silicon Wafer, IEEETrans, on Electron Devices, v. ED-26, pp. 1880-1886; Berg et al., 1994,Micro Total Analysis Systems, New York, Kluwer; Webster et al., 1996,Monolithic Capillary Gel Electrophoresis Stage with On-Chip Detector inInternational Conference On Micro Electromechanical Systems, MEMS 96,pp. 491496; and Mastrangelo et al., 1989, Vacuum-Sealed SiliconMicromachined Incandescent Light Source, in Intl. Electron DevicesMeeting, IDEM 89, pp. 503-506; and other references cited herein andfound in the scientific and patent literature.

Methods of fabrication of complex microfluidic circuits usingelastomeric are known and are described in Unger et al., 2000, Science288:113-116; Quake & Scherer, 2000, “From micro to nanofabrication withsoft materials” Science 290: 1536-40; Xia et al., 1998, “Softlithography” Angewandte Chemie-International Edition 37:551-575; Ungeret al., 2000, “Monolithic microfabricated valves and pumps by multilayersoft lithography” Science 288:113-116; Thorsen et al., 2002,“Microfluidic large-scale integration” Science 298:580-584; Chou et al.,2000, “Microfabricated Rotary Pump” Biomedical Microdevices 3:323-330;Liu et al., 2003, “Solving the “world-to-chip” interface problem with amicrofluidic matrix” Analytical Chemistry 75, 4718-23,” and otherreferences cited herein and known in the art.

Although the present invention has been described in detail withreference to specific embodiments, those of skill in the art willrecognize that modifications and improvements are within the scope andspirit of the invention, as set forth in the claims which follow. Allpublications and patent documents (patents, published patentapplications, and unpublished patent applications) cited herein areincorporated herein by reference as if each such publication or documentwas specifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any such document is pertinent prior art, nor doesit constitute any admission as to the contents or date of the same. Theinvention having now been described by way of written description andexample, those of skill in the art will recognize that the invention canbe practiced in a variety of embodiments and that the foregoingdescription and examples are for purposes of illustration and notlimitation of the following claims.

The invention claimed is:
 1. A method comprising: in a unit cell of amicrofluidic device, said unit cell comprising in a liquid environment:(1) a microfluidic channel having a substrate, a molecular complexcomprising a first molecule immobilized in a contact area of thesubstrate, and a second molecule bound to the first molecule and thusindirectly bound to the substrate, and a movable element that uponactuation contacts the substrate in the contact area wherein the movableelement has a geometry such that when it is fully actuated, the contactarea extends less than the width of the substrate of the microfluidicchannel at the location of the contact area, actuating the movableelement causing it to contact the substrate in the contact area therebyphysically trapping the first and second molecules bound to thesubstrate in the contact area while substantially expelling solvent andsolute molecules; or (2) a microfluidic channel having a substrate, afirst molecule immobilized in a contact area of the substrate, a secondmolecule, a movable element that upon actuation contacts the substratein the contact area, wherein the movable element has a geometry suchthat when it is fully actuated, the contact area extends less than thewidth of the substrate of the microfluidic channel at the location ofthe contact area, actuating the movable element causing it to contactthe substrate in the contact area thereby physically trapping the firstmolecule and any second molecules bound to the first moleculesubstantially expelling solvent and unbound second molecules.
 2. Themethod of claim 1 further comprising de-actuating the movable element.3. The method of claim 1 wherein the second molecule is labeled with afluorescent dye.
 4. The method of claim 1 further comprising detectingthe trapped first and second molecules.
 5. The method of claim 4 furthercomprising detecting a fluorescent signal from said unit cell.
 6. Themethod of claim 1 wherein the movable element is a deflectableelastomeric membrane.
 7. The method of claim 6 wherein the membrane is afree-standing membrane that when fully actuated contacts themicrofluidic channel substrate without contacting the sides of themicrofluidic channel.
 8. The method of claim 7 wherein contact betweenthe membrane and the substrate occurs medially and extends radiallyoutward.
 9. The method of claim 2 further comprising (i) detecting thefirst and any second molecules after de-actuation, and (ii) changing theliquid environment surrounding the contact area.
 10. The method of claim9 wherein prior to said de-actuating, the liquid environment in the unitcell is changed.
 11. The method of claim 1 wherein the first molecule isan antibody and the second molecule is an antigen.
 12. The method ofclaim 1 wherein the first molecule is a protein and the second moleculeis bound by the protein.
 13. The method of claim 1 wherein, prior toactuating the movable element, the molecular complex is contacted with athird molecule and the effect of the third molecule on formation ordissociation of the complex is determined.
 14. The method of claim 1carried out on at least 100 unit cells of a microfluidic device, whereeach of the 100 unit cells comprises a different first molecule.
 15. Themethod of claim 1 carried out on at least 100 unit cells of amicrofluidic device, where each of the 100 unit cells comprises adifferent second molecule.
 16. The method of claim 13 carried out on atleast 100 unit cells of a microfluidic device, where each of the 100unit cells comprises a different third molecule.
 17. The method of claim1, wherein the contact area extends not more than 75% of the width ofthe microfluidic channel substrate at the location of the contact areawhen the movable element is fully actuated.
 18. The method of claim 17,wherein the contact area extends not more than 50% of the width of themicrofluidic channel substrate at the location of the contact area whenthe movable element is fully actuated.
 19. A method comprising: in aunit cell of a microfluidic device, said unit cell comprising in aliquid environment: (1) a microfluidic channel having a substrate, amolecular complex comprising a first molecule immobilized in a contactarea of the substrate, and a second molecule bound to the first moleculeand thus indirectly bound to the substrate, and a movable element thatupon actuation contacts the substrate in the contact area of thesubstrate wherein the movable element when fully actuated does not blockflow of fluid through the microfluidic channel, actuating the movableelement causing it to contact the substrate in the contact area therebyphysically trapping the first and second molecules bound to thesubstrate in the contact area while substantially expelling solvent andsolute molecules; or (2) a microfluidic channel having a substrate, afirst molecule immobilized in a contact area of the substrate, a secondmolecule, a movable element that upon actuation contacts the substratein the contact area of the substrate, wherein the movable element whenfully actuated does not block flow of fluid through the microfluidicchannel, actuating the movable element causing it to contact thesubstrate in the contact area thereby physically trapping the firstmolecule and any second molecules bound to the first moleculesubstantially expelling solvent and unbound second molecules.
 20. Themethod of claim 1, wherein the microfluidic channel is a bulgedmicrofluidic flow channel comprising a widened and rounded portion, andthe contact area is located in said widened and rounded portion.
 21. Themethod of claim 6, wherein the elastomeric membrane has a diametersmaller than the width of the microfluidic channel.
 22. A methodcomprising: in a unit cell of a microfluidic device, said unit cellcomprising in a liquid environment: (1) a microfluidic channel having asubstrate, a molecular complex comprising a first molecule immobilizedin a contact area of the substrate, and a second molecule bound to thefirst molecule and thus indirectly bound to the substrate, and adeflectable membrane that upon actuation contacts the substrate in thecontact area of the substrate, wherein the contact occurs medially andextends radially outward, actuating the deflectable membrane causing itto contact the substrate in the contact area thereby physically trappingthe first and second molecules bound to the substrate in the contactarea while substantially expelling solvent and solute molecules; or (2)a microfluidic channel having a substrate, a first molecule immobilizedin a contact area of the substrate, a second molecule, a deflectablemembrane that upon actuation contacts the substrate in the contact areaof the substrate, wherein the contact occurs medially and extendsradially outward, actuating the deflectable membrane causing it tocontact the substrate in the contact area thereby physically trappingthe first molecule and any second molecules bound to the first moleculesubstantially expelling solvent and unbound second molecules.